Human IgM antibodies, and diagnostic and therapeutic uses thereof particularly in the central nervous system

ABSTRACT

Antibodies, and particularly human antibodies, are disclosed that demonstrate activity in the treatment of demyelinating diseases as well as other diseases of the central nervous system that are of viral, bacterial or idiopathic origin, including neural dysfunction caused by spinal cord injury. Neuromodulatory agents are set forth that include and comprise a material selected from the group consisting of an antibody capable of binding structures or cells in the central nervous system, a peptide analog, a hapten, active fragments thereof, agonists thereof, mimics thereof, monomers thereof and combinations thereof. The neuromodulatory agent has one or more of the following characteristics: it is capable of inducing remyelination; binding to neural tissue; promoting Ca ++  signaling with oligodendrocytes; and promoting cellular proliferation of glial cells. Amino acid and DNA sequences of exemplary antibodies are disclosed. Methods are described for treating demyelinating diseases, and diseases of the central nervous system of humans and domestic animals, using polyclonal IgM antibodies and human monoclonal antibodies sHIgm22(LYM 22), sHIgm46(LYM46) ebvHIgM MSI19D10, CB2bG8, AKJR4, CB2iE12, CB2iE7, MSI19E5 and MSI10E10, active fragments thereof and the like. The invention also extends to the use of human antibodies, fragments, peptide derivatives and like materials, and their use in diagnostic and therapeutic applications, including screening assays for the discovery of additional antibodies that bind to cells of the nervous system, particularly oligodendrocytes.

CROSS REFERENCE TO RELATED APPLICATIONS

The present Application is a Divisional of application Ser. No.12/313,515, filed Nov. 20, 2008 now U.S. Pat. No. 7,807,166, which is acontinuation-in-part of application Ser. No. 10/010,729, filed Nov. 13,2001, and now U.S. Pat. No. 7,473,432, issued Jan. 6, 2009, which is, inturn, a continuation-in-part of application Ser. No. 09/730,473, filedDec. 5, 2000, now abandoned copending application Ser. No. 09/568,531,filed May 10, 2000, application Ser. No. 09/580,787, filed May 30, 2000now abandoned, application Ser. No. 09/322,862, filed May 28, 1999 nowabandoned, application Ser. No. 08/779,784, filed Jan. 7, 1997 nowabandoned, application Ser. No. 08/692,084, filed Aug. 8, 1996 nowabandoned, which is a continuation-in-part of application Ser. No.08/236,520, filed Apr. 29, 1994, and now U.S. Pat. No. 5,591,629, issuedJan. 7, 1997. Applicants claim the benefit of these applications under35 U.S.C. §120, all of which are incorporated herein by reference intheir entireties.

GOVERNMENT SUPPORT

The invention described herein was supported in whole or in part by theNational Institutes of Health, Grant No. NS-24180 and the NationalMultiple Sclerosis Society Grant No. RG-1878-B-2. The United StatesGovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates generally to the field of neurobiology,and more particularly to the identification of autoantibodies that playa role in central nervous system function and therapy. The inventionalso relates to diagnostic and therapeutic materials and methods,including by way of example, pharmaceutical compositions, methods oftreatment of diseases associated with neurological impairment, methodsof regeneration and restoration of neural function, screening assays andvaccines.

BACKGROUND OF THE INVENTION

Multiple sclerosis (MS) is a chronic, frequent progressive, inflammatorycentral nervous system (CNS) disease characterized pathologically byprimary demyelination, usually without initial axonal injury. Theetiology and pathogenesis of MS are unknown. Several immunologicalfeatures of MS, and its moderate association with certain majorhistocompatibility complex alleles, has prompted the speculation that MSis an immune-mediated disease.

An autoimmune hypothesis is supported by the experimental autoimmune(allergic) encephalomyelitis (EAE) model, where injection of certainmyelin components into genetically susceptible animals leads to Tcell-mediated CNS demyelination. However, specific autoantigens andpathogenic myelin-reactive T cells have not been definitively identifiedin the CNS of MS patients, nor is MS associated with other autoimmunediseases. An alternative hypothesis, based upon epidemiological data, isthat an environmental factor, perhaps an unidentified virus,precipitates an inflammatory response in the CNS, which leads to eitherdirect or indirect (“bystander”) myelin destruction, potentially with aninduced autoimmune component. This hypothesis is supported by evidencethat several naturally occurring viral infections, both in humans andanimals, can cause demyelination. One commonly utilized experimentalviral model is induced by Theiler's murine encephalomyelitis virus(TMEV) (Dal Canto, M. C., and Lipton, H. L., Am. J. Path., 88:497-500(1977)).

The limited efficacy of current therapies for MS and other demyelinatingdiseases, has stimulated interest in novel therapies to ameliorate thesediseases. However, due to the apparently complex etiopathogenesis ofthese diseases, potentially involving both environmental and autoimmunefactors, the need still exists for an effective treatment of thesedemyelinating disorders.

In earlier related patent applications referred to hereinabove, a groupof autoantibodies were identified that were found to exhibit activity inthe central nervous system, and that were particularly associated withthe stimulation of remyelination. One of the objectives of theapplicants has been to investigate the full range of activities of theantibodies and concomitantly, to identify other members of the classthat demonstrate such activities. Accordingly, it is toward thefulfillment of the foregoing and other objectives that the presentinvention is directed.

The citation of any reference herein should not be construed as anadmission that such reference is available as “Prior Art” to the instantapplication.

SUMMARY OF THE INVENTION

In a first aspect of the present invention, human autoantibodies,including both polyclonal antibodies and monoclonal antibodies, havebeen identified and isolated, that demonstrate activity in thepromotion, stimulation, regeneration and/or remyelination of neurons inthe central nervous system, and/or in the blocking or reduction ofdemyelination in the central nervous system. Polyclonal IgMimmunoglobulin antibodies have been tested and utilized herein and theinvention extends to polyclonal IgM immunoglobulin, particularly humanpolyclonal IgM immunoglobulin, and its use in the promotion,stimulation, regeneration and/or remyelination of neurons in the centralnervous system, and/or in the blocking or reduction of demyelination inthe central nervous system. Particular antibodies have been identifiedand tested herein, and the invention accordingly extends to humanautoantibodies, which human autoantibodies are exemplified by sHIgM22(LYM 22), sHIgM46 (LYM46), ebvHIgM MSI19D10, CB2bG8, AKJR4, CB2iE12,CB21E7, MSI19E5 and MSI10E10. The invention further provides fragments,monomers, and recombinant antibodies derived from or related to thehuman antibodies of the present invention. Thus, the inventionparticularly extends to fragments, monomers or recombinant antibodiesderived from or based on any of sHIgM22 (LYM 22), sHIgM46 (LYM46),ebvHIgM MSI19D10, CB2bG8, AKJR4, CB2iE12, CB21E7, MSI19E5 and MSI10E10.The invention also provides in another aspect, an assay for screeningother antibodies and related binding partners, including haptens andpeptide analogs, that may exhibit a like therapeutic activity. Suchactivities would include the treatment or prevention of neurologicalinjuries or dysfunctions such as multiple sclerosis, ALS, stroke,Parkinsons disease and Alzheimers disease.

The present invention relates in another aspect to the promotion orstimulation of regeneration or remyelination of central nervous systemaxons in a mammal. Specifically, the present invention relates tomethods of stimulating the remyelination of central nervous system (CNS)axons using autoantibodies, and particularly human autoantibodies,including antibodies of the IgM subtype and monomers thereof, ormixtures and/or active fragments thereof, characterized by their abilityto bind to structures and cells in the central nervous system,particularly including oligodendrocytes. Certain of these monoclonals(mAbs) are referred to herein as SCH94.03, SCH 79.08, O1, O4, A2B5, andO9. Of these monoclonal antibodies, O1, O4, O9, A2B5 and HNK-1 arewell-known oligodendrocyte-reactive (OL-reactive) monoclonal antibodies.See, for instance, Eisenbarth et al., Proc. Natl. Acad. Sci. USA, 76(1979), 4913-4917, and Abo et al. J. Immunol., 127 (1981), 1024-1029).The monoclonal antibodies referred to as SCH94.03 and SCH 79.08, and thecorresponding hybridomas producing them, have been deposited on Apr. 28,1994, and Feb. 27, 1996, respectively, under the terms of the BudapestTreaty, with the American Type Culture Collection (ATCC) and given ATCCAccession Nos. CRL 11627 and HB12057, respectively. The presentinvention also extends to the preparation and use of humanautoantibodies, which human autoantibodies are exemplified by sHIgM22(LYM 22), sHIgM46 (LYM46), ebvHIgM MSI19D10, CB2bG8, AKJR4, CB2iE12,CB21E7, MSI19E5 and MSI10E10. The heavy and light chain variable regionsequences of these antibodies are provided herein as follows: LYM 22 areset forth in FIGS. 35 and 36; the corresponding sequences for ebvHIgMMSI19D10 are set forth in FIGS. 37 and 38; the corresponding sequencesfor CB2bG8 are set forth in FIGS. 45 and 46; the corresponding sequencesfor AKJR4 are set forth in FIGS. 55 and 56; the corresponding sequencesfor CB2iE12 are set forth in FIGS. 57 and 58; the correspondingsequences for CB21E7 are set forth in FIGS. 59 and 60; the correspondinglight chain sequences for MSI19E5 are set forth in FIG. 61; thecorresponding heavy and light chain sequences for sHIgM46 (LYM46) areset forth in FIGS. 71 and 72; and accordingly, the invention extends toantibodies and corresponding antibody proteins, and small molecules suchas haptens, that have or correspond at least in part to the sequencesset forth in the noted Figures.

This work provides confirmation of the generic utility of the group ofnatural autoantibodies as effective in producing remyelination of thecentral nervous system. In accordance with a further embodiment of theinvention, and as stated above, a broader class of antibodies has beendefined and is disclosed herein. Specifically, human polyclonal andmonoclonal autoantibodies are disclosed and prepared in accordanceherewith, that provide affinity for neural tissue and both diagnosticand therapeutic capability. The invention extends further in that thenewly identified antibodies may be employed for a variety of purposessuch as the promotion of remyelination, regeneration of damaged nervecells, neuronal protection, neuronal outgrowth and the like.

A significant feature and advantage of the present invention resides inthe source of the antibodies, as they may be obtained directly from thehost or patient, and then used to promote safer self-therapies. Morebroadly, the development of peptides, small molecules and the like,based on these endogenous materials reduces, if not eliminates, possiblepathologies or dysfunctions such as autoimmune reactions, that mayresult from the in vivo introduction and use of exogenous materials.Also, the endogenous origin of the antibodies offers a further advantagein that it may be possible to study the repair process in the patient orhost, and potentially identify an underlying mechanism of action in thetreatment of the condition, that itself may yield further therapeuticinsights and strategies.

Moreover, the identification of the relationship between agents thatpromote calcium signaling, as by the induction of Ca⁺⁺ peaks, onoligodendrocytes, and the initiation and/or promotion of the notedtherapeutic activities, is contemplated to provide a method ofidentifying therapeutic agents by the demonstration of calcium signalingon glial cells, e.g. oligodendrocytes and astrocytes. Accordingly, theinvention extends to this use and activity as well.

The antibodies described herein may be used to screen peptide librariesor haptens whereby the reactive peptides or haptens can then be isolatedand tested for their ability to remyelinate, block or reducedemyelination, induce cellular proliferation, differentiation, neuraloutgrowth, neurite sprouting, Ca⁺⁺ signaling and/or block cell death,e.g. hydrogen-peroxide induced cell death. Once isolated and purified,such peptides can then be used to screen for other polyclonal ormonoclonal antibodies or other molecules that may induce remyelination,cellular proliferation or differentiation, neuronal outgrowth, neuritesprouting and/or Ca⁺⁺ signaling, the last mentioned noted herein to berelevant to the proliferation and the corresponding activity of glialcells. Particularly, peptides, haptens, and other moleculescorresponding to the antibodies of the invention may be identified bytheir ability to bind to oligodendrocytes and thereby inducing neuralrehabilitation, such as remyelination, regeneration and neuroprotection.

The invention is also broadly directed to peptides which bind to theautoantibodies described herein, whereby these peptides by virtue oftheir sequence, three-dimensional structure, or conformational changesarising from antibody binding, can be used in and of themselves aspeptide vaccines. In a further aspect of the invention, these peptidesmay have neuromodulatory and/or immunomodulatory properties and mayprovide a method of inducing a neural cell proliferative response and/orneuroprotective, neuroregenerative and/or remyelinating role in mammalsin need of such therapy.

Likewise, the invention includes haptens that may bind to the peptides,the antibodies and/or other relevant substrates and that may possessimmunogenicity, so that they may also function as active components intherapeutic formulations, also including vaccines. In a particularembodiment, one or more haptens may be combined with other of thepeptides of the present invention, in a vaccine formulation.

In yet a further aspect of the invention these peptides can beformulated as pharmaceutical compositions with stabilizers to preventproteolytic degradation, thus extending their half-life to be givenorally, subcutaneously, intravenously, intranasally, intrathecally or asliposome preparations to mammals in need of such therapy.

The present invention also relates to methods of treating demyelinatingdiseases in mammals, such as multiple sclerosis in humans, and viraldiseases of the central nervous system of humans and domestic animals,such as post-infectious encephalomyelitis, or prophylacticallyinhibiting the initiation or progression of demyelination in thesedisease states, using the monoclonal antibodies, or active fragmentsthereof, of this invention. This invention further relates to in vitromethods of producing and stimulating the proliferation of glial cells,such as oligodendrocytes, and the use of these glial cells to treatdemyelinating diseases.

In a further aspect, the invention extends to a group of molecules thatwill be referred to herein as neuromodulatory agents, and that arenotable in their therapeutic activity in the CNS. Accordingly, theinvention relates to neuromodulatory agents with particulareffectiveness in the CNS, which agents comprise a material selected fromthe group consisting of an antibody of the IgM subtype, a peptideanalog, a hapten, active fragments thereof, monomers thereof, agoniststhereof, mimics thereof, and combinations thereof. Related antibodies ofdifferent subtypes, including those that have undergone a class switch(naturally or as generated by recombinant or synthetic means), are alsocontemplated, wherein the class switch antibodies have characteristic asneuromodulatory agents useful in the methods of the present invention.The neuromodulatory agents have one or more of the followingcharacteristics: they induce remyelination and/or cellular proliferationof glial cells; and/or evoke Ca⁺⁺ signaling with oligodendrocytes;and/or block cell death, e.g. hydrogen-peroxide induced cell death.

The antibodies of and for use in the present invention includepolyclonal antibodies, and the invention particularly providespolyclonal IgM antibodies, particularly polyclonal IgM immunoglobulinand preparation thereof, more particularly pooled polyclonal IgMimmunoglobulin, particularly preferred pooled polyclonal human IgMimmunoglobulin.

More particularly, the antibodies comprehended within the scope ofneuromodulatory agents of the invention may be selected from the groupconsisting of mAb SCH94.03, SCH79.08, O1, O4, O9, A2B5, HNK-1, sHIgM22(LYM 22), ebvHIgM MSI19D10, sHIgM46 (LYM46), CB2bG8, AKJR4, CB2iE12,CB21E7, MSI19E5, MSI10E10, mixtures thereof, monomers thereof, activefragments thereof, and natural or synthetic autoantibodies having thecharacteristics of the particular mAb SCH94.03, SCH79.08, O1, O4, O9,A2B5, HNK-1, sHIgM22 (LYM 22), ebvHIgM MSI19D10,sHIgM46 (LYM46), CB2bG8,AKJR4, CB2iE12, CB21E7, MSI19E5 and MSI10E10. Antibodies furthercomprehended within the scope of the neuromodulatory agents of theinvention are recombinant antibodies derived from mAb SCH94.03,SCH79.08, O1, O4, O9, A2B5, HNK-1, sHIgM22 (LYM 22), ebvHIgM MSI19D10,sHIgM46 (LYM46), CB2bG8, AKJR4, CB2iE12, CB21E7, and MSI10E10. Thepresent neuromodulatory agents may be derived from mammalian cells andspecifically, may be derived from human cells. Further, theneuromodulatory agents may comprise a polypeptide having an amino acidsequence corresponding at least in part, to a sequence selected from thegroup consisting of FIG. 35 (SEQ ID NO: 8, 7), FIG. 36 (SEQ ID NO: 10,9), FIG. 37 (SEQ ID NO: 11, 12), FIG. 38 (SEQ ID NO: 13, 14), FIG. 45(SEQ ID NO: 15, 16), FIG. 46 (SEQ ID NO: 17, 18), FIG. 55 (SEQ ID NO:25, 26), FIG. 56 (SEQ ID NO: 27, 28), FIG. 57 (SEQ ID NO: 29, 30), FIG.58 (SEQ ID NO: 31, 32), FIG. 59 (SEQ ID NO: 33, 34), FIG. 60 (SEQ ID NO:35, 36), FIG. 61 (SEQ ID NO: 37, 38), FIG. 71 (SEQ ID NO: 49), FIG. 72(SEQ ID NO: 51) and active fragments thereof. Recombinant or syntheticantibodies derived or based therefrom and corresponding at least in partto a sequence selected from the above group are further included in thepresent invention.

The present invention thus relates to the monoclonal antibody sHIgM22(LYM22), monomers thereof, active fragments thereof, and natural orsynthetic antibodies having the characteristics of sHIgM22. Recombinantantibodies derived from sHIgM22 are further contemplated and areprovided herein. An sHIgM22(LYM22) antibody myeloma has been depositedas ATTC Accession No. PTA-8671. The invention provides antibodiescomprising a polypeptide having an amino acid sequence corresponding atleast in part to a sequence selected from FIG. 35 (SEQ ID NO: 8, 7) andFIG. 36 (SEQ ID NO: 10, 9), and active fragments thereof. Recombinant orsynthetic antibodies derived or based therefrom and corresponding atleast in part to a sequence selected from SEQ ID NO: 8, 7, 10 and 9 arefurther included in the present invention.

The present invention further relates to the monoclonal antibody sHIgM46(LYM46), monomers thereof, active fragments thereof, and natural orsynthetic antibodies having the characteristics of sHIgM46. Recombinantantibodies derived from sHIgM46 are further contemplated and areprovided herein. The invention provides antibodies comprising apolypeptide having an amino acid sequence corresponding at least in partto a sequence selected from FIG. 71 (SEQ ID NO: 49) and FIG. 72 (SEQ IDNO: 51), and active fragments thereof. Recombinant or syntheticantibodies derived or based therefrom and corresponding at least in partto a sequence selected from SEQ ID NO: 49 and 51 are further included inthe present invention.

The present invention further relates to sequences identified for mouseantibodies suitable and useful in the present invention asneuromodulatory agents having one or more of the followingcharacteristics: they induce remyelination and/or cellular proliferationof glial cells; and/or evoke Ca⁺⁺ signaling with oligodendrocytes. Inparticular, antibody sequences are provided in FIGS. 67-70. Thus, theneuromodulatory agents of the present invention may comprise apolypeptide having an amino acid sequence corresponding at least inpart, to a sequence selected from the group consisting of FIG. 67 (SEQID NO: 41, 42), FIG. 68 (SEQ ID NO: 43, 44), FIG. 69 (SEQ ID NO: 45,46), FIG. 70 (SEQ ID NO: 47, 48), and active fragments thereof.Recombinant or synthetic antibodies derived or based therefrom andcorresponding at least in part to a sequence selected from the abovegroup are further included in the present invention.

The present invention also relates to a recombinant DNA molecule orcloned gene, or a degenerate variant thereof, which encodes a class ofmolecules that will also be referred to herein as neuromodulatoryagents, and that include and may be selected from the antibodies of theinvention, and particularly antibodies having sequences corresponding atleast in part, to the sequences presented in FIGS. 35-38, 45, 46 and55-61 and FIGS. 67-72; peptides that may correspond at least in part tothe antibodies of the present invention, that will also be referred toherein as antibody peptides, and for example, peptides having one ormore sequences corresponding at least in part to FIGS. 35-38, 45, 46 and55-61 and FIGS. 67-72; and small molecules such as haptens; includingrecombinant DNA molecules or cloned genes having the same orcomplementary sequences.

More particularly, the recombinant DNA molecule comprises a DNA sequenceor degenerate variant thereof, which encodes an antibody, a peptideanalog thereof, a hapten corresponding thereto, or an active fragmentthereof, and which may be selected from the group consisting of:

-   -   (A) the DNA sequence encoding a protein having a sequence        corresponding to at least a portion of FIG. 35 (SEQ ID NO: 8,        7);    -   (B) the DNA sequence encoding a protein having a sequence        corresponding to at least a portion of FIG. 36 (SEQ ID NO: 10,        9);    -   (C) the DNA sequence encoding a protein having a sequence        corresponding to at least a portion of FIG. 37 (SEQ ID NO: 11,        12);    -   (D) the DNA sequence encoding a protein having a sequence        corresponding to at least a portion of FIG. 38 (SEQ ID NO: 13,        14);    -   (E) the DNA sequence encoding a protein having a sequence        corresponding to at least a portion of FIG. 45 (SEQ ID NO: 15,        16);    -   (F) the DNA sequence encoding a protein having a sequence        corresponding to at least a portion of FIG. 46 (SEQ ID NO: 17,        18);    -   (G) the DNA sequence encoding a protein having a sequence        corresponding to at least a portion of FIG. 55 (SEQ ID NO: 25,        26);    -   (H) the DNA sequence encoding a protein having a sequence        corresponding to at least a portion of FIG. 56 (SEQ ID NO: 27,        28);    -   (I) the DNA sequence encoding a protein having a sequence        corresponding to at least a portion of FIG. 57 (SEQ ID NO: 29,        30);    -   (J) the DNA sequence encoding a protein having a sequence        corresponding to at least a portion of FIG. 58 (SEQ ID NO: 31,        32);    -   (K) the DNA sequence encoding a protein having a sequence        corresponding to at least a portion of FIG. 59 (SEQ ID NO: 33,        34);    -   (L) the DNA sequence encoding a protein having a sequence        corresponding to at least a portion of FIG. 60 (SEQ ID NO: 35,        36);    -   (M) the DNA sequence encoding a protein having a sequence        corresponding to at least a portion of FIG. 61 (SEQ ID NO: 37,        38);    -   (N) the DNA sequence encoding a protein having a sequence        corresponding to at least a portion of FIG. 67 (SEQ ID NO: 41,        42);    -   (O) the DNA sequence encoding a protein having a sequence        corresponding to at least a portion of FIG. 68 (SEQ ID NO: 43,        44);    -   (P) the DNA sequence encoding a protein having a sequence        corresponding to at least a portion of FIG. 69 (SEQ ID NO: 45,        46);    -   (Q) the DNA sequence encoding a protein having a sequence        corresponding to at least a portion of FIG. 70 (SEQ ID NO: 47,        48);    -   (R) the DNA sequence encoding a protein having a sequence        corresponding to at least a portion of FIG. 71 (SEQ ID NO: 49,        50);    -   (S) the DNA sequence encoding a protein having a sequence        corresponding to at least a portion of FIG. 72 (SEQ ID NO: 51,        52);    -   (T) DNA sequences that hybridize to any of the foregoing DNA        sequences under standard hybridization conditions; and    -   (U) DNA sequences that code on expression for an amino acid        sequence encoded by any of the foregoing DNA sequences.

The present invention also includes proteins derived from orcorresponding to said antibodies, or fragments or derivatives thereof,having the activities noted herein, and that display the amino acidsequences set forth and described above and selected at least in part,from the group consisting of FIGS. 35-38, 45, 46 and 55-61 and FIGS.67-72. The present invention likewise extends to haptens thatdemonstrate the same activities as the proteins or antibody peptides,and that may be administered for therapeutic purposes in like fashion,as by formulation in a vaccine. In one embodiment, a vaccine includingboth peptides and haptens may be prepared.

In a further embodiment of the invention, the full DNA sequence of therecombinant DNA molecule or cloned gene so determined may be operativelylinked to an expression control sequence which may be introduced into anappropriate host. The invention accordingly extends to unicellular hoststransformed with the cloned gene or recombinant DNA molecule comprisinga DNA sequence encoding the present antibody peptides.

In a particular embodiment, the variable region DNA sequence of anantibody of the present invention may be utilized in generatingsynthetic antibody(ies). In particular, variable region sequence may becombined with its natural or a genetically provided constant regionsequence to provide a synthetic antibody. The present invention providesvectors for generating synthetic antibodies derived from and comprisingthe DNA sequences, particularly variable region sequences, of theantibodies of the present invention.

According to other preferred features of certain preferred embodimentsof the present invention, a recombinant expression system is provided toproduce biologically active animal or particularly human antibodypeptides.

The present invention includes several means for the preparation ofclones of the autoantibodies, peptides, corresponding haptens, or othersmall molecule analogs thereof, including as illustrated herein knownrecombinant techniques, and the invention is accordingly intended tocover such synthetic preparations within its scope. The isolation of thecDNA and amino acid sequences disclosed herein facilitates thereproduction of the present antibodies or their analogs by suchrecombinant techniques, and accordingly, the invention extends toexpression vectors prepared from the disclosed DNA sequences forexpression in host systems by recombinant DNA techniques, and to theresulting transformed hosts.

The invention includes an assay system for screening of potential drugseffective to modulate the neurological activity of target mammalianneural cells by, for example, potentiating the activity of the presentautoantibodies or their analogs. In one instance, the test drug could beadministered to a cellular sample with the ligand that suppresses orinhibits the activity of the autoantibodies, or an extract containingthe suppressed antibodies, to determine its effect upon the bindingactivity of the autoantibodies to any chemical sample (including DNA),or to the test drug, by comparison with a control.

The assay system could more importantly be adapted to identify drugs orother entities that are capable of binding to the autoantibodies and/ortheir targets, including peptides, haptens, other factors or proteins,whether found in the cytoplasm, the nucleus or elsewhere, therebypotentiating antibody activity, including e.g. immune response, neuralgrowth, neuroprotection and remyelination, and the correspondingtherapeutic activities noted herein. Such assay would be useful in theidentification of drug candidates from among peptide and other smallmolecule libraries, sera, and other relevant body fluids, and in thedevelopment of drugs that would be specific either in the promotion orthe inhibition of particular cellular activity, or that would potentiatesuch activity, in time or in level of activity. For example, such drugsmight be used to promote remyelination, or to treat other pathologies orinjuries, as for example, in making CNS neurons able or better able toengage in regrowth or regeneration.

The present invention likewise extends to the development of antibodiescorresponding to the neuromodulatory agents of the invention, includingnaturally raised and recombinantly prepared antibodies. For example, theantibodies could be used to screen expression libraries to obtain thegene or genes that encode the peptides that may function asneuromodulatory agents, and that could function e.g. in a vaccine. Suchantibodies could include both polyclonal and monoclonal antibodiesprepared by known genetic techniques, as well as bi-specific (chimeric)antibodies, and antibodies including other functionalities suiting themfor additional diagnostic use conjunctive with their capability ofemulating or modulating the activity of the human autoantibodies thatare a part of the neuromodulatory agents of the present invention.

Thus, the neuromodulatory agents, their analogs and/or analogs, and anyantagonists or antibodies that may be raised thereto, are capable of usein connection with various diagnostic techniques, includingimmunoassays, such as a radioimmunoassay, using for example, an antibodyto the neuromodulatory agents that has been labeled by eitherradioactive addition, or radioiodination.

In an immunoassay, a control quantity of the antagonists or antibodiesthereto, or the like may be prepared and labeled with an enzyme, aspecific binding partner and/or a radioactive element, and may then beintroduced into a cellular sample. After the labeled material or itsbinding partner(s) has had an opportunity to react with sites within thesample, the resulting mass may be examined by known techniques, whichmay vary with the nature of the label attached.

In the instance where a radioactive label, such as the isotopes ³H, ¹⁴C,³²P, ³⁵S, ³⁶Cl, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁹⁰Y, ¹²⁵I, ¹³¹I, and ¹⁸⁶Re areused, known currently available counting procedures may be utilized. Inthe instance where the label is an enzyme, detection may be accomplishedby any of the presently utilized colorimetric, spectrophotometric,fluorospectrophotometric, amperometric or gasometric techniques known inthe art.

The present invention includes an assay system which may be prepared inthe form of a test kit for the quantitative analysis of the extent ofthe presence of the neuromodulatory agents, or to identify drugs orother agents that may mimic or block their activity. The system or testkit may comprise a labeled component prepared by one of the radioactiveand/or enzymatic techniques discussed herein, coupling a label to theneuromodulatory agents, their agonists and/or antagonists, and one ormore additional immunochemical reagents, at least one of which is a freeor immobilized ligand, capable either of binding with the labeledcomponent, its binding partner, one of the components to be determinedor their binding partner(s).

In a particular and further aspect, the present invention extends to theuse and application of the antibodies of the present invention,particularly autoantibodies, including antibodies of the IgM subtype andmonomers thereof, or mixtures and/or active fragments thereof,characterized by their ability to bind to structures and cells in thecentral nervous system, particularly including oligodendrocytes, inimaging and in vivo diagnostic applications. Thus, the antibodies, byvirtue of their ability to bind to structures and cells in the centralnervous system, particularly including oligodendrocytes, can be utilizedvia immunofluorescent, radioactive and other diagnostically suitabletags as imaging agents or imaging molecules for the characterization ofthe nervous system, including the central nervous system and thediagnosis, monitoring and assessment of nervous disease, particularlyincluding multiple sclerosis. The antibodies may further be utilized asimaging agents or imaging molecules in the diagnosis, monitoring andassessment of stroke, spinal cord injury and various dementias includingAlzheimer's disease.

In a further embodiment, the present invention relates to certaintherapeutic methods which would be based upon the activity of theneuromodulatory agents, their subunits, or active fragments thereof,peptide equivalents thereof, analogs thereof, or upon agents or otherdrugs determined to possess the same activity. A first therapeuticmethod is associated with the prevention of the manifestations ofconditions causally related to or following from the binding activity ofthe antibodies or their subunits, and comprises administering an agentcapable of stimulating the production and/or activity of theneuromodulatory agents, the corresponding autoantibodies, antibodypeptides, active fragments or subunits thereof, either individually orin mixture with each other in an amount effective to prevent or treatthe development of those conditions in the host. For example, drugs orother binding partners to the antibodies or their fragments, or thelike, may be administered to potentiate neuroregenerative and/orneuroprotective activity, or to stimulate remyelination as in thetreatment of multiple sclerosis.

More specifically, the therapeutic method generally referred to hereincould include the method for the treatment of various pathologies orother cellular dysfunctions and derangements by the administration ofpharmaceutical compositions that may comprise effective inhibitors orenhancers of activation of the neuromodulatory agents, or other equallyeffective drugs developed for instance by a drug screening assayprepared and used in accordance with an aspect of the present inventiondiscussed above. For example, drugs or other binding partners to theneuromodulatory agents or like proteins, having sequences correspondingat least in part to the sequences as represented by FIG. 35 (SEQ ID NO:8, 7), FIG. 36 (SEQ ID NO: 10, 9), FIG. 37 (SEQ ID NO: 11, 12), FIG. 38(SEQ ID NO: 13, 14), FIG. 45 (SEQ ID NO: 15, 16), FIG. 46 (SEQ ID NO:17, 18), FIG. 55 (SEQ ID NO: 25, 26), FIG. 56 (SEQ ID NO: 27, 28), FIG.57 (SEQ ID NO: 29, 30), FIG. 58 (SEQ ID NO: 31, 32), FIG. 59 (SEQ ID NO:33, 34), FIG. 60 (SEQ ID NO: 35, 36), FIG. 61 (SEQ ID NO: 37, 38), FIG.71 (SEQ ID NO: 49, 50), FIG. 72 (SEQ ID NO: 51, 52) may be administeredto inhibit or potentiate neuroregeneration, neuroprotection, orremyelination, as in the treatment of Parkinsons disease or multiplesclerosis. In particular, the proteins of one or more antibodiesselected from the group of sHIgM22 (LYM22), ebvHIgM MSI19D10, sHIgM46(LYM46), CB2bG8, AKJR4, CB2iE12, CB21E7 and MSI19E5, whose sequences arepresented in FIGS. 35-38, 45, 46, 55-61, and 71-72, their antibodies,agonists, antagonists, monomers or active fragments thereof, includingmixtures and combinations thereof, could be prepared in pharmaceuticalformulations including vaccines, for administration in instances whereinneuroregenerative and/or neuroprotective therapy or remyelination isappropriate, such as to treat Alzheimers disease, ALS, Parkinsonsdisease, or spinal cord injury. The present invention includescombinations or mixtures of the antibodies provided herein, wherein morethan one of the antibodies, particularly human antibodies, mostparticularly selected from the group of sHIgM22, sHIgM46, MSI19E10,CB2bG8, AKJR4, CB2iE12, CB21E7, MSI19E5, and MSI10E10 can be prepared inpharmaceutical and therapeutic compositions or formulations.Combinations or mixtures of various human antibodies, mouse antibodies,or monomers, fragments, recombinant or synthetic antibodies derivedtherefrom or based thereon are also provided by and included in thepresent invention. The human antibodies (extending to monomers,fragments, recombinant or synthetic antibodies derived therefrom) areparticularly selected from the group of sHIgM22, sHIgM46, MSI19E10,CB2bG8, AKJR4, CB2iE12, CB21E7, MSI19E5, and MSI10E10. The mouseantibodies (extending to monomers, fragments, recombinant or syntheticantibodies and humanized antibodies derived therefrom) are particularlyselected from the group of SCH 94.03, SCH79.08, O1, O4, O9, A2B5 andHNK-1. In addition, the invention provides further combinations of theantibody(ies) with therapeutic compounds, drugs or agents useful in anysuch neuroregenerative and/or neuroprotective therapy or remyelination.For instance, the antibody formulation or composition of the presentinvention may be combined with therapeutic compounds for the treatmentof multiple sclerosis, including but not limited to beta interferonformulations (Betaseron, etc.) and coploymer 1 (Copaxone).

Accordingly, it is a principal object of the present invention toprovide neuromodulatory agents, including the human autoantibodies andcorresponding antibody peptides, haptens, analogs and active fragmentsthereof in purified form that exhibits certain characteristics andactivities associated with the promotion of neuroregenerative and/orneuroprotective activity.

It is a further object of the present invention to provide a method fordetecting the presence, amount and activity of the autoantibodies inmammals in which invasive, spontaneous, or idiopathic pathologicalstates are suspected to be present.

It is a further object of the present invention to provide a method andassociated assay system for screening substances such as drugs, agentsand the like, potentially effective in either mimicking the activity orcombating any adverse effects of the autoantibodies and/or theirfragments, subunits or the like, in mammals.

It is thus an object of the present invention to provide methods fortreating demyelinating diseases in mammals, such as multiple sclerosisin humans, and viral diseases of the central nervous system of humansand domestic animals, such as post-infectious encephalomyelitis, orprophylactically inhibiting the initiation or progression ofdemyelination in these disease states, using the described monoclonalautoantibodies, active fragments thereof, or other natural or syntheticautoantibodies having the characteristics of mAb SCH94.03, SCH 79.08,O1, O4, O9, A2B5, HNK-1, and the human autoantibodies exemplified bysHIgM22 (LYM 22), ebvHIgM MSI19D10, sHIgM46 (LYM46), CB2bG8, AKJR4,CB2iE12, CB21E7, MSI19E5 and MSI10E10.

It is further an object of the present invention to provide in vitromethods of producing, and stimulating the proliferation of, glial cells,such as oligodendrocytes, and the use of these glial cells to treatdemyelinating diseases.

It is a still further object of the present invention to provide thepresent neuromodulatory agents, and pharmaceutical compositions,including vaccines comprising the same, for use in therapeutic methodswhich comprise or are based upon the present neuromodulatory agents, andparticularly the human autoantibodies, fragments, including peptidefragments, haptens, subunits, agonists, binding partner(s), or uponagents or drugs that control the production, or that mimic or antagonizethe activities of the neuromodulatory agents.

It is a still further object of the present invention to provide assaymethods including screening assays, for the identification of drugs andother molecules that mimic or antagonize the neuromodulatory agents ofthe invention, and that can consequently be considered for use astherapeutic agents.

Other objects and advantages will become apparent to those skilled inthe art from a review of the following description which proceeds withreference to the following illustrative drawings.

DESCRIPTION OF THE FIGURES

FIG. 1 is a graph depicting the dose-response characteristics ofantibody-mediated proliferation of cells in mixed rat brain culture.

FIG. 2 is a graph depicting the temporal profile of antibody-mediatedproliferation of cells in mixed rat brain culture.

FIG. 3A-3D shows light and electron micrographs of CNS remyelinationpromoted by mAb SCH94.03. (A) Light micrograph of spinal cord sectionfrom a chronically infected SJL/J mouse treated with SCH94.03 showingCNS remyelination. (B) Light micrograph of spinal cord section from achronically infected SJL/J muse treated with a control IgM showingextensive demyelination, and the relative absence of remyelination.Inflammatory cells, including macrophages with ingested myelin debrisare indicated by arrows. The asterisk indicates a representative nakedaxon. (C) Light micrograph of spinal cord section with normal myelin.(D) Electron micrograph of spinal cord section from an animal treatedwith SCH94.03 showing multiple axons with abnormally thin myelin sheathsrelative to axon diameter. The star in the upper right-hand cornerindicates an axon with normal myelin sheath thickness. Arrowheads pointto astrocytic processes, which are intimately associated withremyelinated axons. Scale bars represent 13 μm in A-C, and 2 μm in D.

FIG. 4 is a graph depicting the correlation between the change inclinical disease and morphological remyelination.

FIG. 5 is a graph depicting the dose-response relationship betweentreatment with mAb SCH94.03 and CNS remyelination. Area of CNAremyelination (●) and percentage of lesion area with remyelination (∘)in animals treated with various doses of mAb SCH94.03.

FIG. 6 shows a Western blot of TMEV proteins. Lysates from infected L2fibroblast cells were separated by SDS-PAGE, transferred tonitrocellulose, and blotted with SCH94.03 (lane 1), SCH93.32 (lane 2),serum from susceptible mice chronically infected with TMEV (lane 3), andpolyclonal rabbit anti-TMEV IgG (lane 4). Molecular weights areindicated on the left in kilodaltons (kDa). The position andidentification of the major TMEV capsid proteins are indicated on theright.

FIGS. 7A-7D shows the immunostaining of cultured glial cells and frozenCNS tissue sections with mAb SCH94.03. Scale bars represent 15 μm.

FIGS. 8A-8C shows the results of SCH94.03 (FIG. 8A) and control IgMs(FIGS. 8B and 8C) binding to protein antigens as determined by ELISA.

FIG. 9 shows the results of SCH94.03 F(ab2)′ binding to protein antigensas determined by ELISA.

FIG. 10A-10C show the results of SCH94.03 (FIG. 10A) and control IgMs(FIGS. 10B and 10C) binding to chemical haptens as determined by ELISA.

FIGS. 11A and 11B shows the alignment of the immunoglobulin light (FIG.11A, SEQ ID NO: 63, 64) and heavy (FIG. 11B, SEQ ID NO: 65, 66) chainvariable region sequences of SCH94.03 and control IgM, CH12, andgermline Ig gene segments.

FIG. 12 shows the nucleotide and deduced amino acid sequences of V_(H),D and J_(H) regions encoding O1, compared with the unrearranged V_(H)segment transcript A1 and A4, and the JH germline gene (SEQ. ID NO: 1,67). Dashed lines indicate identity with unrearranged V_(H) segmenttranscript A1 and A4. Underline indicates identity with germline AP2gene family (DSP2.3, 2.4, 2.6). Amino acids are represented by thesingle-letter code. CDR represents the complementarity determiningregion. This sequence has been assigned the GenBank TM/EMBL Data BankAccession number L41877.

FIG. 13 shows the nucleotide and deduced amino acid sequences of V_(H),D and J_(H) regions encoding O4 and HNK-1 (SEQ. ID NO: 2, 68), comparedwith those reported for germline gene V_(H)101 and J_(H), and fornatural autoantibody D23. Dashed lines indicate identity with V_(H)101and J_(H)4. Underline indicates identity with germline DFL16.1. Aminoacids are represented by the single-letter code. CDR represents thecomplementarity determining region. These sequences have been assignedthe GenBank TM/EMBL Data Bank Accession Numbers L41878 (O4) and L41876(HNK-a).

FIG. 14 shows the nucleotide and deduced amino acid sequences of V_(H),D and J_(H) regions encoding A2B5 (SEQ. ID NO: 3, 69), compared withthose reported for germline gene V1 and J_(H)3 germline gene. Dashedlines indicate identity with germline gene V1 and J_(H)3. Underlineindicates identity with germline DFL16.2. Amino acids are represented bythe single-letter code. CDR represents the complementarity determiningregion. This sequence has been assigned the GenBank TM/EMBL Data BankAccession Number L41874.

FIG. 15 shows the nucleotide and deduced amino acid sequences of V_(H)and J_(H) regions encoding O1 and O4 (SEQ. ID NO: 4, 70), compared withthose reported for myeloma MOPC21, for natural autoantibody E7 and for3_(x)2 germline gene. Dashed lines indicate identity with MOPC21 andgermline gene J_(H)2 (N, undetermined nucleotide). Amino acids arerepresented by the single-letter code. CDR represents thecomplementarity determining region. These sequence have been assignedthe GenBank TM/EMBL Data Bank Accession Numbers L41879 (O1) and L41881(O4).

FIG. 16 shows the nucleotide and deduced amino acid sequences of V_(H)and J_(H) regions encoding HNK-1 (SEQ. ID NO: 5, 71), compared withthose reported for germline V_(H)41, myeloma MOPC21, and J_(H)2. Dashedlines indicate identity with germline genes. Amino acids are representedby the single-letter code. CDR represents the complementaritydetermining region. This sequence has been assigned the GenBank TM/EMBLData Bank Accession Number L41880.

FIG. 17 shows the nucleotide and deduced amino acid sequences of V_(H)and J_(H) regions encoding A2B5 (SEQ ID NO: 6, 72). Dashed linesindicate identity with germline J_(H). Amino acids are represented bythe single-letter code. CDR represents the complementarity determiningregion. This sequence has been assigned the GenBank TM/EMBL Data BankAccession Number L41875.

FIG. 18 is a graph showing the reactivity of O1, O4, A2B5 and control(TEPC183 and XXMEN-OES)IgMχ mAbs by direct ELISA.

FIG. 19 comprises photographs showing that polyclonal human antibodiesand serum-derived human monoclonal IgM antibodies (sHIgMs) bind withhigh specificity to surface antigens on cells in slices of cerebellum.Indirect immunofluorescent labeling of unfixed slices of postnatal ratcerebellum. sHIgMs demonstrate a variety of specificities to cellpopulations and structures within an unfixed brain slice. This propertywas used as one of the criteria to select candidate antibodies to testin vivo for the ability to promote remyelination (see Table 7 and FIG.12). Polyclonal human IgG binds very weakly to many structures withinthe cerebellum, including white matter and Purkinje cells (A), whilepolyclonal human IgM strongly binds to myelin and presumptiveoligodendrocytes within the central white matter of the folia, Purkinjecell bodies and many small cells within the granular and molecular layer(B). sHIgM22 (C) binds well to the cytoskeleton of damaged astrocytesoverlying the central white matter of the folia, Purkinje cells andtheir dendritic arborizations, and to small round cells in the molecularlayer. sHIgM22 weakly, but uniformly, labels the surface of granulecells. sHIgM 14 (D) binds well to cells of the granular layer andPurkinje cells located at the surface of the slice, while the centralwhite matter of the folia is largely devoid of label. sHIgM 1 (E) labelsthe cytoskeleton of astrocytes overlying the central white matter of thefolia. All other structures are identified just above background levels.sHIgM 2 (F) binds to cells of the granular layer and to fiberstraversing the central white matter of the folia. Magnification x.

FIG. 20 comprises photographs showing that additional sHIgMs bind withhigh specificity to cells in slices of cerebellum. Indirectimmunofluorescent labeling of unfixed slices of postnatal ratcerebellum. sHIgM demonstrate a variety of specificities to cellpopulations and structures within an unfixed brain slice. sHIgM 12 (A)binds to lend a spongy appearance to the central white matter of thefolia, and a uniform label over the molecular layer, reminiscent of anextracellular matrix molecule. Overlying astrocytes are also welldefined. sHIgM 29 (B) binds weakly to many structures in the cerebellumwith an intensity just above background, except for a small populationof neurons in the granular and molecular layer. Axon extensions over 100(m long are clearly delineated. sHIgM 31 (C) and sHIgM 50 (F) each bindpredominately to the granular layer, with little binding to the whitematter, Purkinje cells or astrocytes. The binding pattern of sHIgM 50 isalso reminiscent of an extracellular matrix molecule. sHIgM 42 (D) bindsin a fibrous pattern to the entire folia, molecular and granular layersand white matter. sHIgM 46 (E) binds in a fibrous pattern to thegranular layer and white matter. The Purkinje cell bodies are welldefined.

FIG. 21 comprise photographs that show that sHIgMs bind with highspecificity to unfixed slices of adult human cortical white matter.Indirect immunofluorescent labeling of unfixed slices of adult humancortical white matter. Cortical human white matter was obtained atautopsy from an individual with no CNS infection or trauma. The cause ofdeath was other than CNS-related. Tissue was obtained on ice andmaintained cold throughout the antibody labeling procedure. sHIgM 2 (A)binds to only a few cells within the field of view. In contrast, otherssHIgMs bind human white matter quite well and with a high degree ofspecificity. sHIgM 32 binds to type 2 astrocyte-appearing cells (C),while sHIgM 31 binds to many unidentified round cell bodies (B). sHIgM26 binds to oligodendrocyte-appearing cells and fibrous white matter(E). sHIgM22 binds to human cortical white matter in a manner that issuggestive of an extracellular matrix bound molecule (D). Magnificationx.

FIG. 22 comprise photographs that show that EBV-immortalized humanB-cell clone-derived monoclonal IgM antibodies (ebvHIgMs) bind with highspecificity to surface antigens on cells in the cerebellum. Indirectimmunofluorescent labeling of unfixed slices of postnatal ratcerebellum. ebvHIgMs demonstrate a variety of specificities to cellpopulations and structures within an unfixed rat brain slice. ebvHIgMMSI19E15 (A) binds to fibrous structures within the white matter and tothe granular and molecular layer in a pattern of near confluency.ebvHIgM AKJR4 (B) binds almost exclusively to the granular layer. Smallcells within the molecular layer are also identified. ebvHIgMs MSI17A2(C) and MSI20H10 (E) bind to the central white matter, the granular andmolecular layer and Purkinje cells with varying degrees of intensity.ebvHIgM MSI16E6 (D) demonstrates a very strong affinity for Purkinjecells and their dendrictic arbors, while the granular layer is far lessdistinctly labeled. ebvHIgM MSI7E11 (F) binds in a punctate manner toonly a few glial-appearing cells at the surface of the brain slice.Magnification x.

FIG. 23 shows that additional ebvHIgMs that bind with high specificityto surface antigens on cells in slices of cerebellum. Indirectimmunofluorescent labeling of unfixed slices of postnatal ratcerebellum. ebvHIgMs demonstrate a variety of specificities to cellpopulations and structures within an unfixed brain slice. Each panelshows the terminal end of a single cerebellar folia, including thecentral white matter, and the granular, Purkinje and molecular layers.Supernatants containing ebvHIgMs were incubated 1:1 with buffered mediaon slices of brain. Many ebvHIgMs bind to white matter, Purkinje cellbodies, and small cells within the molecular layer, but with varyingaffinities. ebvHIgM MSI19D10 (A) binds strongly to cells of the granularlayer and to Purkinje cells and their dendritic arbors, in addition toweakly identifying white matter and astrocytes. ebvHIgM MSI19D10 wastested for the ability to promote remyelination in vivo (see Table 7 andFIG. 13). Other brain-binding ebvHIgMs, CB2bG8(B), CB2eC2 (C), CB2iE12(D), and MSI10E10 (F) have been isolated and warrant further study, buthave not been tested in vivo. CB2eC2 (E) is the typical intensity of anon-reactive Supernatants. Magnification x.

FIG. 24 shows that polyclonal human IgM binds to oligodendrocytes inculture. By immunocytochemistry, polyclonal human IgM stains the surfaceof a subpopulation of oligodendrocytes. No reactivity to oligodendrocytesurface antigens was observed with polyclonal human IgG, or sera fromsHIgM 1 or SHIgM 2. Immunocytochemistry with pooled human IgM or IgG infixed and permeabilized cells showed minimal staining of intracellularstructures.

FIG. 25 shows that sHIgMs bind with high specificity to surface antigenson glial cells in culture. Indirect immunofluorescent labeling of liverat primary mixed glial cell cultures at nine days post seeding. sHIgMsdemonstrate a variety of specificities as to the cell types bound aswell as the cell differentiation-stage identified in mixed glialcultures. sHIgM 12 binds to clusters of presumptive oligodendrocyteprogenitors (A, green label) in the midst of more mature O4+oligodendrocytes (A, red label). sHIgM22 binds to mature stages ofoligodendrocytes (B) adherent to the surface of the glial culture. sHIgM46 strongly binds to both mature stages of oligodendrocyte (C, center offigure) and immature stages of oligodendrocyte with a fainter, punctatelabel (C, left side of figure). sHIgM 42 (D) and sHIgM 51 (F) both bindto mature stages of the oligodendrocyte and faintly, the underlyingastrocytes. sHIgM 30 binds to the cell bodies of most cells in theculture, while no process extensions are delineated (E). Magnificationx.

FIG. 26 shows that sHIgMs bind to cells of the oligodendrocyte lineagein slices of cerebellum and cultures of mixed primary glial cells. Cellsidentified by sHIgMs co-label with markers for the oligodendrocytelineage. Cells that bind sHIgM22 in an unfixed slice of neonatal ratcerebellum co-label the Rip antibody, a cytoplasmic marker for maturestages of the oligodendrocyte. Double label confocal images demonstratesHIgM22 positive cells (A) that are also Rip positive (C). Images (A)and (C) are merged in (E). Cells that bind sHIgM 51 in mixed primary ratglial cell cultures (B) are also O4 positive (D). O4, an anti-sulfatide,is a well established marker for the oligodendrocyte lineage thatappears prior the cessation of proliferation and is maintained on intothe adult myelin sheath. Images (B) and (D) are merged in (F).Magnification x.

FIG. 27 shows that ebvHIgMs bind to cells of the oligodendrocyte lineagein slices of cerebellum. Cells identified by ebvHIgM MSI19E5 in anunfixed slice of neonatal rat cerebellum co-label with the O4 antibody,an anti-sulfatide and cell-surface marker for oligodendrocytes. Doublelabel confocal images of cells within the white matter of the foliademonstrate ebvHIgM MSI19E5 positive cells (A) that are also O4 positive(B). Images (A) and (B) are merged in (C).

FIG. 28 presents the results of screening sHIgMs for binding to CNSantigens found in spinal cord homogenate. sHIgMs were screened for theirbinding to spinal cord homogenate bound to polystyrene plates. Most ofantigens that bind to the plate are lipids and proteins from the whitematter of the spinal cord. Thus, strong antibody binding to SCHhomogenate may be interpreted as binding to white matter components.Only 1 sHIgM binds to SCH with an OD greater than 1, sHIgM22. Thisantibody also binds well to brain slices, oligodendrocytes in cultureand has been tested for the ability to promote remyeliation in vivo (seeTable 7). This simple assay has proven to be a powerful tool inpredicting the capacity of an antibody to promote remyelination in vivo.Other sHIgMs that bind well to SCH (such as 38 and 49) are under study.

FIG. 29 shows the results of screening ebvHIgMs for binding CNS antigensfound in spinal cord homogenate. ebvHIgMs were screened for theirbinding to spinal cord homogenate bound to polystyrene. Four ebvHIgMsbound to SCH homogenate with a OD greater than 1. One of these, MSI19D10has been tested for the ability to promote remyelination in vivo. (seeTable 7). A low binding antibody, AKJR4 has also been tested in vivo(see Table 7). One other strong binding antibody, AKJR8 is under study.The clones CB2iH 1 and CB 1bD2, produce very little antibody in culture.Again, this simple assay has proven to be a very powerful tool forscreening antibodies, and predicting which antibodies are capable ofpromoting remyelination in vivo.

FIG. 30 demonstrates that polyclonal human antibodies and a sHIgMpromote remyelination in TMEV infected mice. Light photomicrographs ofregions of myelin pathology in the spinal cords of SJL/J micechronically infected with TMEV. Extensive CNS remyelination,characterized by thin myelin sheaths in relation to axon diameter, isobserved in mice after treatment with polyclonal human IgG (A),polyclonal human IgM (B), and sHIgM22(C). Demyelination withoutsignificant remyelination was observed in mice treated with sHIgM 14(D), sHIgM 1(E) and sHIgM 2. Aradite embedded sections were stained with1% p-phenylenediamine. Magnification x. Polyclonal human IgM proved tobe superior in the ability to promote remyelination in vivo thanpolyclonal human IgG (Table 7). Strong CNS specificity appears to be oneof the requirements for an antibody to promote remyelination in vivo,but alone is not sufficient to predict an antibody's capacity to promoteremyelination.

FIG. 31 shows that an ebvHIgM can promote remyelination in TMEV infectedmice. Light photomicrographs of regions of myelin pathology in thespinal cords of SJL/J mice chronically infected with TMEV. Extensive CNSremyelination, characterized by thin myelin sheaths in relation to axondiameter, is observed in mice after treatment with ebvHIgM MSI19D10 (A).Demyelination without significant remyelination was observed in micetreated with ebvHIgM AKJR4 (B). Aradite embedded sections were stainedwith 1% p-phenylenediamine. Again, strong CNS specificity appears to beone of the requirements for an antibody to promote remyelination invivo, but alone is not sufficient to predict an antibody's capacity topromote remyelination.

FIG. 32 presents the quantitation of myelinated axons in lysolecithinlesions treated with human polyclonal IgM. Remyelinated axons/mm2 intreated vs untreated lysolecithin lesions. There are significantly moremyelinated axons in lysolecithin lesions treated with polyclonal humanIgM than animals treated with polyclonal human IgG (p<0.05). One animalin the PBS control group spontaneously remyelinated and thus thedifference between the human antibody treated groups and the controlgroup is not statistically significant (p>0.05).

FIG. 33 demonstrates that human antibodies are polyreactive to chemicalhaptens via ELISA. Antigen binding specificities of immunoglobulinsassessed by direct ELISA. Chemical hapten reactivities of polyclonalhuman IgM, polyclonal human IgG. Abbreviations used in these figures:NP, (4-hydroxy-3-nitrophenyl)acetyl; PhoX, phenyloxazolone; TMA,azophenyltrimethylammonium; FITC, fluorescein; PC,azophenylphosphoryl-choline; ARS, azophenylarsonate; TNP, trinytrophenylacetyl.

FIG. 34 shows that human antibodies are polyreactive to self protein viaELISA. Protein antigen binding specificities of immunoglobulins assessedby direct ELISA. Abbreviations used in these figures: MBP, myelin basicprotein; KLH, keyhole limpet hemocyanin; HEL, hen egg lysozyme; BSA,bovine serum albumin; Rbt, rabbit; Bo, bovine; Mo Hb, mouse hemoglobin.

FIG. 35 presents the sHIgM22 heavy chain variable region amino acid andnucleic acid sequences (SEQ. ID NO: 7, 8, respectively). The sequence isaligned according to the numbering system of human V_(H) sequences inthe publication: Sequences of Proteins of Immunological Interest, Vol I,Fifth Edition (1991), Kabat E.A., Wu, T.T., Perry, H.M. Gottesman, K.S.and Foeller, C., NIH Publication. The sHIgM22 V_(H) is a member of theV_(H) subgroup III. Underlined amino acids have been confirmed byprotein sequencing. Amino acid sequence corresponds to sHIgM22nucleotide sequence. SHIgM22 V_(H) type A and B nucleic acid sequences(SEQ ID NO: 8) are represented only with nucleotides that differ fromthe IGHV3-30/3-30-05*01, IGHJ4*02 and IGHD2-21*02 germline sequences(SEQ ID NO: 81). Two amino acid replacements in the protein sequence ofsHIgM22 V_(H) type B are printed in bold. The sequences of both SHIgM22V_(H) type A and B most closely matched the IGHV3-30/3-30-5*01 germlinesequence (96% homology). References for germline sequences: IMGT, theinternational ImMunoGeneTics database [http://imgt.cnusc.fr:8104].(Initiator and coordinator: Marie-Paule Lefranc, Montpellier, France)

FIG. 36 presents the sHIgM22 light chain variable region amino acid andnucleic acid sequences (SEQ. ID NO: 9, 10, respectively) The sequence isaligned according to the numbering system of human V_(H) sequences inthe publication: Sequences of Proteins of Immunological Interest, Vol I,Fifth Edition (1991), Kabat E.A., Wu, T.T., Perry, H.M. Gottesman, K.S.and Foeller, C., NIH Publication. V_(λ) sHIgM22 is a member of thelambda subgroup I. Underlined amino acids have been confirmed by proteinsequencing. Amino acid sequence corresponds to sHIgM22 nucleotidesequence. SHIgM22 V_(λ) type I and II nucleic acid sequences (SEQ ID NO:10) are represented only with nucleotides that differ from theIGLV1-51*01 and IGLJ3*01 germline sequences (SEQ ID NO: 82). Two aminoacid replacements in the protein sequence of sHIgM22 V_(λ) type II areprinted in bold. The V_(λ) sequences from SHIgM22 most closely matchedthe IGLV-51*01 germline sequence (97% homology). The two genes differfrom their common ancestor by a single nucleotide change. References forgermline sequences: IMGT, the international ImMunoGeneTics database[http://imgt.cnusc.fr:8104]. (Initiator and coordinator: Marie-PauleLefranc, Montpellier, France).

FIG. 37 presents the ebvHIgM MSI19D10 heavy chain variable regionsequence_(SEQ. ID NO: 11, 12).

FIG. 38 presents the ebvHIgM MSI19D10 light chain variable regionsequence (SEQ. ID NO: 13, 14).

FIG. 39 demonstrates that monoclonal antibodies that promoteremyelination cause Ca²⁺ flux in glial cells in culture. The threepanels demonstrate glial Ca²⁺ responses to four different antibodies:two which promote remyelination in vivo, sHIgM22 (A) and SCH94.03 (B),and two which do not promote remyelination, sHIgM 14 (panel C) and CH12(C). Cells which responded exhibited one of two different types ofcalcium spikes, either a fast spike immediately upon addition ofantibody (A & B, red traces), or a broader spike which appears with ashort delay after addition of antibody (A & B, black traces). The smallcolored triangles on the time axis represent the moment antibody (orionophore) were added. Antibodies sHIgM22 and SCH94.03 elicited bothtypes of responses but from different subsets of glial cells (panels A &B). Antibodies sHIgM14 and CH12, which do not promote remyelination invivo, were not observed to cause calcium flux in cultured glia (panelC). At the end of each experiment the calcium ionophore Br-A23187 wasadded to each culture as a control for cellular integrity. Addition ofionophore to viable cells causes a large Ca²⁺ influx which is apparentin each of the experiments that are depicted.

FIG. 40 demonstrates that sHIgMs and ebvHIgMs bind to primary neurons inculture. Indirect immunofluorescent labeling of live primary rat granulecells at six days in culture. sHIgM 12 binds to virtually all axon anddendritic extensions of cerebellar granule cells in culture (A). Thebinding pattern is similar to that observed with anti-gangliosideantibodies, such as mouse antibody A2B5. A2B5 has been shown to promoteremyelination in vivo (Asakara et al, 1998). ebvHIgM CB2iE12 binds onlyto granule cell bodies and their proximal axon extensions (B). Theantigen recognized by CB2iE12 is developmentally regulated, for granulecells in culture are negative for CB2iE12 staining until 4-5 days afterplating. Magnification x.

FIG. 41 demonstrates that mouse monoclonal antibody SCH94.03 binds tothe surface of granule cells in culture. Indirect immunofluorescentlabeling and confocal serial imaging demonstrates that the mousemonoclonal antibody SCH94.03 binds only to the surface in granule cellneurons in culture. The series of images were taken 1 um apart andclearly show the concentric circular rings expected of an externallylabeled spherical cell body with process extensions.

FIG. 42 presents the methodology used to quantify white matter, whitematter pathology and remyelination in the spinal cords of TMEV-infectedmice. Light photomicrograph of a thorasic level spinal cord section froman SJL/J mouse chronically infected with TMEV and treated withpolyclonal human IgM (A). White matter at the periphery stains darkerthan the lighter central gray matter. The area of total white matter istraced (indicated by the red outlines), at a magnification of 40×. Thenat a magnification of 100× the areas of white matter pathology aretraced (indicated by the green outlines). In this example, the areas ofwhite matter pathology appear as lighter areas at the periphery of thesection. Finally, at a magnification of 250× the areas of OLremyelination (indicated by the blues outlines) and SC remyelination(indicated by the yellow outline) are traced. OL remyelination ischaracterized by thin myelin sheaths in relation to axon diameter. Thepercent area of white matter pathology is calculated by dividing thearea in green by the area in red ×100. The percent area of OLremyelination is calculated by dividing the area in blue by the area ingreen ×100. Ten spinal cord cross sections are traced for each animalconsidered and the areas combined to calculate a score for that animal.Generally, 7-8 animals are treated in each experimental group to allowfor deaths and animals that do not contain at least 5% total whitematter pathology. Usually 4-5 treated animals meet the criteria forinclusion into the final data set. A high magnification field of thedorsal column white matter (B, from the area indicated by the asteriskin A) demonstrates significant OL remyelination (arrow). Scale bars are250 μm in A and 20 μm in B.

FIG. 43 Following treatment with human Abs, chronically TMEV-infectedmice demonstrate significant OL remyelination. Light photomicrographs ofrepresentative areas of spinal cord white matter pathology of differenttreatment groups. Treatment with IVIg resulted in significant OLremyelination (A). Almost complete OL remyelination, characterized bydensely packed thin myelin sheaths in relation to axon diameter (B,arrowhead), was observed in sections from the spinal cords of micefollowing treatment with polyclonal human IgM (B) and human mAbs sHIgM22(F) and sHIgM46 (G). In contrast, following treatment with human mAbssHIgM1 (C), sHIgM2 (D), sHIgM14 (E) or PBS (H) mice demonstrated whitematter pathology without significant OL remyelination. Infiltratinginflammatory cells and macrophages ingesting myelin debris (A,arrowhead), signs of active myelin destruction were also evident. Spinalcord cross sections in four of eight animals treated with sHIgM22 andfive of five animals treated with sHIgM46 contained at least one area ofnearly confluent OL remyelination, a rare event indicating significanttissue repair. In contrast, the 10 spinal cord cross sections from eachmouse treated with sHIgM1, sHIgM2, sHIgM14, or PBS contained none. Scalebar is 20 mm.

FIG. 44 Human mAbs isolated for their ability to bind to rat OLs alsobind to the surface of human OLs in culture. sHIgM14 (A), which did notpromote remyelination, and sHIgM22 (B) and sHIgM46 (C), which didpromote remyelination, bound to the perikaryon and elaborate process andmembrane extensions of sulfatide positive human OLs maintained inculture for 3 weeks. sHIgM2 (D, green channel) is an example of a humanmAb that did not bind to sulfatide positive (D, red channel) human OLs.Nuclei are labeled blue. IVIg, polyclonal human IgM, and human mAbssHIgM1 and sHIgM2 did not bind to the surface of human OLs at any timepoint examined. Scale bar is 25 mm.

FIG. 45 presents the heavy chain variable region sequence of EBVtransformant antibody CB2b-G8 (SEQ. ID NO: 15, 16).

FIG. 46 presents the light chain variable region sequence of EBVtransformant antibody CB2b-G8 (SEQ. ID NO: 17, 18).

FIG. 47 Amplification of light chain RNA and protein expression intransfected hybridoma cells by methotrexate amplification of adHfR-containing expression plasmid. Expression plasmid containing thecoding sequence for humanized 94.03 kappa light chain under control ofthe CMV promoter along with a linked dHfR gene under control of the SV40promoter was introduced by electroporation into the immunoglobulinnegative F3B6 human/mouse hybridoma cell line. Cell under minimalmethotrexate selection (0.5 μg/ml) and those that had undergone morestringent selection (51.2 μg/ml) were cultured to harvest supernatant toassess light chain secretion and RNA to assess light chain geneexpression. Northern blot analysis indicates substantial amplificationof RNA expression in one clone (#5). Protein expression was increasedfollowing methotrexate selection in clone 4, but not in clone 5. Thesefindings indicate that methotrexate amplification sometimes results inthe amplification of mRNA and protein expression by closely linkedgenes, but in other cases no amplification of transcription and proteinsynthesis is seen.

FIG. 48 Amplifiable vectors encoding humanized 94.03 and sHIgM22. Toppanel is the prototype vector containing the coding sequence forhumanized 94.03 light chain (κ) and a hybrid genomic construct encodinghumanized 94.03 heavy chain (μ). Bottom panel is a similar constructthat contains the coding sequences derived from the sHIgM22 sequence.Both of these constructs have been expressed in F3B6 hybridoma cellsunder minimal methotrexate selection conditions and are now beingselected under more stringent conditions to isolate clones expressingamplified amount of immunoglobulin.

FIG. 49 Postnatal Rat cerebellum stained with murine and humanized94.03. Cerebellum sections were stained with mouse and humanized 94.03.Bound antibody was localized using fluorescent secondary antibodyreagent specific for mouse or human IgM, respectively. Both antibodiesshowed similar staining patterns to white matter tracks and astrocytesin the cerebellum.

FIG. 50 Isolation of an IgG variant of 94.03. A natural switch variantof 94.03 was isolated from culture by sorting for cells expressing IgGon their surface. Pre-sort and post-sort profiles of the cell culturesare shown. IgG cells were isolated from the post-sort population bylimiting dilution cloning. The antibody produced was identified as IgG1using IgG isotype specific antibodies.

FIG. 51 Demonstration that the IgG1 producing cells were in fact avariant of 94.03. RNA was isolated from the clonal IgG1 expressingcells. cDNA was generated using RTPCR with primers specific for thevariable region of 94.03 and the constant region of the γ1 isotype. Theresulting DNA was sequenced to demonstrate the precise splicing junctionexpected for a spontaneous switch variant.

FIG. 52 presents the heavy chain variable region sequence of mouse O9antibody_(SEQ. ID NO: 19, 20).

FIG. 53 presents the kappa light chain 1 variable region sequence ofmouse O9 variable region sequence of mouse O9 antibody (SEQ. ID NO: 21,22).

FIG. 54 presents the kappa light chain 2 variable region sequence ofmouse O9 antibody (SEQ. ID NO: 23, 24).

FIG. 55 presents the AKJR4 heavy chain variable region sequence (SEQ. IDNO: 25, 26).

FIG. 56 presents the AKJR4 kappa light chain variable region sequence(SEQ. ID NO: 27, 28).

FIG. 57 presents the CB2iE12 heavy chain variable region sequence (SEQ.ID NO: 29, 30).

FIG. 58 presents the CB2iE12 kappa light chain variable region sequence(SEQ. ID NO: 31, 32).

FIG. 59 presents the CB21E7 heavy chain variable region sequence (SEQ.ID NO: 33, 34).

FIG. 60 presents the CB21E7 kappa light chain variable region sequence(SEQ. ID NO: 35, 36).

FIG. 61 presents the MSI 19E5 light chain variable region sequence (SEQ.ID NO: 37, 38).

FIG. 62 presents the kappa light chain 2 of the mouse O4 antibody (SEQ.ID NO: 39, 40).

FIG. 63A depicts assessment of glial cell proliferation by mouseantibody 94.03.

FIG. 63B depicts assessment of glial cell proliferation by humanantibodies lym22, recombinant 22 and lym5.

FIG. 64 depicts assessment of glial cell proliferation by humanantibodies native 22, 22BII, AKJR4 and AKJR8.

FIG. 65 depicts assessment of glial cell proliferation by mouse antibodyO9 versus serum Ig of SJL mice.

FIG. 66 depicts immunofluorescence assessment of white matter binding bysHIgM22 and RsHIgM22.

FIG. 67 depicts the kappa light chain sequence of antibody O4_(SEQ. IDNO: 41, 42).

FIG. 68 depicts the kappa light chain sequence of antibody O1 (SEQ. IDNO: 43, 44).

FIG. 69 depicts the kappa light chain sequence of antibody HNK-1 (SEQ.ID NO: 45, 46).

FIG. 70 depicts the kappa light chain sequence of antibody A2B5 (SEQ. IDNO: 47, 48).

FIG. 71 depicts the Lym 46 heavy chain sequence (SEQ. ID NO: 49, 50).

FIG. 72 depicts the Lym 46 kappa light chain sequence (SEQ. ID NO: 51,52).

FIG. 73 depicts vector pAD46M for expression of recombinant Lym 46.

FIG. 74 depicts vector pUD22BIIM for expression of recombinant Lym22,containing the DHFR gene for amplification of the vector withmethotrexate.

FIG. 75 depicts vector pUD46M for expression of recombinant Lym46,containing the DHFR gene for amplification of the vector withmethotrexate.

FIG. 76 depicts vector pAD22G1/G2 for generation of and recombinantexpression of IgG subclass G1 and G2 of Lym 22.

FIG. 77 depicts vector pUD22G1/G2 for generation of and recombinantexpression of IgG subclass G1 and G2 of Lym 22, also containing the DHFRgene for amplification of the vector with methotrexate.

FIG. 78 depicts vector pAD46G1/G2 for generation of and recombinantexpression of IgG subclass G1 and G2 of Lym 46.

FIG. 79 depicts vector pUD46G1/G2 for generation of and recombinantexpression of IgG subclass G1 and G2 of Lym 46, also containing the DHFRgene for amplification of the vector with methotrexate.

FIG. 80 depicts assessment of percent demyelination and inflammation inTMEV infected SJL mice treated with ●PBS, ▪SHIgM22, and ▴SHIgM46 21 dayspost infection.

FIG. 81 depicts assessment of percent demyelination in TMEV infected SJLmice treated with SHIgM46 or SHIgM22.

FIG. 82 depicts assessment of percent demyelination in TMEV infected SJLmice treated with SHIgM46 versus all other remyelination promotingmonoclonal antibodies.

FIG. 83 presents internalization of labeled ⁴⁵Ca in undifferentiated CG4oligodendrocyte cells. ⁴⁵Ca internalization is presented for untreatedcells and cells treated with Lym22, Lym2, 94.03 IgM and 94.03 IgG,respectively.

FIG. 84 presents internalization of labeled ⁴⁵Ca in CG4 oligodendrocytecells for untreated cells and cells treated with Lym22, 94.03, Lym46,and BDNF, respectively.

FIG. 85 presents a hydrogen peroxide (H₂0₂) kill curve—the percentage ofGC4 cells surviving—on exposure to 0.00 mM, 0.01 mM, 0.10 mM and 1.0 mMH₂0₂, in the absence and presence of Lym22 antibody.

FIG. 86 presents assessment and comparison of H₂0₂—induced cell death byMTT assay or cell number in untreated, 94.03 IgM treated, H₂0₂ treatedand H₂0₂+94.03 IgM treated GC4 cells.

DETAILED DESCRIPTION

The present invention relates to the promotion, stimulation,regeneration, protection, and/or remyelination of central nervous systemaxons in a mammal. Specifically, the present invention relates tomethods of stimulating the remyelination of central nervous system (CNS)axons using a monoclonal autoantibody, including of the IgM subtype andmonomers thereof, particularly including human antibodies, or an activefragment thereof, characterized by its ability to bind to structures andcells of the central nervous system, particularly oligodendrocytes, or anatural or synthetic analog thereof. The pres using a monoclonalautoantibody, including of the IgM subtype and monomers thereof,particularly including human antibodies, or an active fragment thereof,characterized by its ability to bind to structures and cells of thecentral nervous system, particularly oligodendrocytes, or a natural orsynthetic analog thereof.

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook et al, “Molecular Cloning:A Laboratory Manual” (1989); “Current Protocols in Molecular Biology”Volumes I-III [Ausubel, R. M., ed. (1994)]; “Cell Biology: A LaboratoryHandbook” Volumes I-III [J. E. Celis, ed. (1994))]; “Current Protocolsin Immunology” Volumes I-III [Coligan, J. E., ed. (1994)];“Oligonucleotide Synthesis” (M. J. Gait ed. 1984); “Nucleic AcidHybridization” [B. D. Hames & S. J. Higgins eds. (1985)]; “TranscriptionAnd Translation” [B. D. Hames & S. J. Higgins, eds. (1984)]; “AnimalCell Culture” [R. I. Freshney, ed. (1986)]; “Immobilized Cells AndEnzymes” [IRL Press, (1986)]; B. Perbal, “A Practical Guide To MolecularCloning” (1984).

Therefore, if appearing herein, the following terms shall have thedefinitions set out below.

The term “neuromodulatory agent(s)” as used herein singularly throughoutthe present application and claims, is intended to refer to a broadclass of materials that function to promote neurite outgrowth,regeneration and remyelination with particular benefit and effect in theCNS, and therefore includes the antibodies of the IgM sub-type, andparticularly, human antibodies such as those referred to specificallyherein as sHIgM22 (LYM 22), ebvHIgM MSI19D10, sHIgM46 (LYM46), CB2bG8,AKJR4, CB2iE12, CB21E7 and MSI19E5, peptide analogs, haptens, activefragments thereof, monomers thereof, agonists, mimics and the like,including such materials as may have at least partial sequencesimilarity to the peptide sequences set forth in FIGS. 35-38, 45, 46,55-61 and 71-72. An sHIgM22(LYM22) antibody myeloma has been depositedas ATT′C Accession No. PTA-8671. Neuromodulatory agent(s) also includesand encompasses combinations or mixtures of more than one of theantibodies provided herein, including monomers or active fragmentsthereof.

Also, the terms “neuromodulatory agent,” “autoantibody,” “antibodypeptide,” “peptide,” “hapten” and any variants not specifically listed,may be used herein interchangeably, to the extent that they may allrefer to and include proteinaceous material including single or multipleproteins, and extends to those proteins having the amino acid sequencedata described herein and presented in FIGS. 35-38, 45, 46, 55-61 and71-72 (SEQ ID NOS: 7, 8, 10, 9, 11, 12, 13, 14, 15, 16, 17, 18, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 49 and 51), and theprofile of activities set forth herein and in the Claims. Accordingly,proteins displaying substantially equivalent or altered activity arelikewise contemplated. These modifications may be deliberate, forexample, such as modifications obtained through site-directedmutagenesis, or may be accidental, such as those obtained throughmutations in hosts that are producers of the complex or its namedsubunits. Also, the terms “neuromodulatory agent,” “autoantibody,”“antibody peptide,” “peptide,” “hapten” are intended where appropriate,to include within their scope proteins specifically recited herein aswell as all substantially homologous analogs and allelic variations.

The amino acid residues described herein are preferred to be in the “L”isomeric form. However, residues in the “D” isomeric form can besubstituted for any L-amino acid residue, as long as the desiredfunctional property of immunoglobulin-binding is retained by thepolypeptide. NH₂ refers to the free amino group present at the aminoterminus of a polypeptide. COOH refers to the free carboxy group presentat the carboxy terminus of a polypeptide. In keeping with standardpolypeptide nomenclature, J. Biol. Chem., 243:3552-59 (1969),abbreviations for amino acid residues are shown in the following Tableof Correspondence:

TABLE OF CORRESPONDENCE SYMBOL 1-Letter 3-Letter AMINO ACID Y Tyrtyrosine G Gly glycine F Phe phenylalanine M Met methionine A Alaalanine S Ser serine I Ile isoleucine L Leu leucine T Thr threonine VVal valine P Pro proline K Lys lysine H His histidine Q Gln glutamine EGln glutamic acid W Trp tryptophan R Arg arginine D Asp aspartic acid NAsn aspargine C Cys cysteine

It should be noted that all amino-acid residue sequences are representedherein by formulae whose left and right orientation is in theconventional direction of amino-terminus to carboxy-terminus.Furthermore, it should be noted that a dash at the beginning or end ofan amino acid residue sequence indicates a peptide bond to a furthersequence of one or more amino-acid residues. The above Table ispresented to correlate the three-letter and one-letter notations whichmay appear alternately herein.

A “replicon” is any genetic element (e.g., plasmid, chromosome, virus)that functions as an autonomous unit of DNA replication in vivo; i.e.,capable of replication under its own control.

A “vector” is a replicon, such as plasmid, phage or cosmid, to whichanother DNA segment may be attached so as to bring about the replicationof the attached segment.

A “DNA molecule” refers to the polymeric form of deoxyribonucleotides(adenine, guanine, thymine, or cytosine) in its either single strandedform, or a double-stranded helix. This term refers only to the primaryand secondary structure of the molecule, and does not limit it to anyparticular tertiary forms. Thus, this term includes double-stranded DNAfound, inter alia, in linear DNA molecules (e.g., restrictionfragments), viruses, plasmids, and chromosomes. In discussing thestructure of particular double-stranded DNA molecules, sequences may bedescribed herein according to the normal convention of giving only thesequence in the 5′ to 3′ direction along the nontranscribed strand ofDNA (i.e., the strand having a sequence homologous to the mRNA).

An “origin of replication” refers to those DNA sequences thatparticipate in DNA synthesis.

A DNA “coding sequence” is a double-stranded DNA sequence which istranscribed and translated into a polypeptide in vivo when placed underthe control of appropriate regulatory sequences. The boundaries of thecoding sequence are determined by a start codon at the 5′ (amino)terminus and a translation stop codon at the 3′ (carboxyl) terminus. Acoding sequence can include, but is not limited to, prokaryoticsequences, cDNA from eukaryotic mRNA, genomic DNA sequences fromeukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. Apolyadenylation signal and transcription termination sequence willusually be located 3′ to the coding sequence.

Transcriptional and translational control sequences are DNA regulatorysequences, such as promoters, enhancers, polyadenylation signals,terminators, and the like, that provide for the expression of a codingsequence in a host cell.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence is bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site (convenientlydefined by mapping with nuclease S1), as well as protein binding domains(consensus sequences) responsible for the binding of RNA polymerase.Eukaryotic promoters will often, but not always, contain “TATA” boxesand “CAT” boxes. Prokaryotic promoters contain Shine-Dalgarno sequencesin addition to the −10 and −35 consensus sequences.

An “expression control sequence” is a DNA sequence that controls andregulates the transcription and translation of another DNA sequence. Acoding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then translated intothe protein encoded by the coding sequence.

A “signal sequence” can be included before the coding sequence. Thissequence encodes a signal peptide, N-terminal to the polypeptide, thatcommunicates to the host cell to direct the polypeptide to the cellsurface or secrete the polypeptide into the media, and this signalpeptide is clipped off by the host cell before the protein leaves thecell. Signal sequences can be found associated with a variety ofproteins native to prokaryotes and eukaryotes.

The term “oligonucleotide,” as used herein in referring to probes of thepresent invention, is defined as a molecule comprised of two or moreribonucleotides, preferably more than three. Its exact size will dependupon many factors which, in turn, depend upon the ultimate function anduse of the oligonucleotide.

The term “primer” as used herein refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product, which is complementary to a nucleic acid strand, isinduced, i.e., in the presence of nucleotides and an inducing agent suchas a DNA polymerase and at a suitable temperature and pH. The primer maybe either single-stranded or double-stranded and must be sufficientlylong to prime the synthesis of the desired extension product in thepresence of the inducing agent. The exact length of the primer willdepend upon many factors, including temperature, source of primer anduse of the method. For example, for diagnostic applications, dependingon the complexity of the target sequence, the oligonucleotide primertypically contains 15-25 or more nucleotides, although it may containfewer nucleotides.

The primers herein are selected to be “substantially” complementary todifferent strands of a particular target DNA sequence. This means thatthe primers must be sufficiently complementary to hybridize with theirrespective strands. Therefore, the primer sequence need not reflect theexact sequence of the template. For example, a non-complementarynucleotide fragment may be attached to the 5′ end of the primer, withthe remainder of the primer sequence being complementary to the strand.Alternatively, non-complementary bases or longer sequences can beinterspersed into the primer, provided that the primer sequence hassufficient complementarity with the sequence of the strand to hybridizetherewith and thereby form the template for the synthesis of theextension product.

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes, each of which cut double-strandedDNA at or near a specific nucleotide sequence.

A cell has been “transformed” by exogenous or heterologous DNA when suchDNA has been introduced inside the cell. The transforming DNA may or maynot be integrated (covalently linked) into chromosomal DNA making up thegenome of the cell. In prokaryotes, yeast, and mammalian cells forexample, the transforming DNA may be maintained on an episomal elementsuch as a plasmid. With respect to eukaryotic cells, a stablytransformed cell is one in which the transforming DNA has becomeintegrated into a chromosome so that it is inherited by daughter cellsthrough chromosome replication. This stability is demonstrated by theability of the eukaryotic cell to establish cell lines or clonescomprised of a population of daughter cells containing the transformingDNA. A “clone” is a population of cells derived from a single cell orcommon ancestor by mitosis. A “cell line” is a clone of a primary cellthat is capable of stable growth in vitro for many generations.

Two DNA sequences are “substantially homologous” when at least about 75%(preferably at least about 80%, and most preferably at least about 90 or95%) of the nucleotides match over the defined length of the DNAsequences. Sequences that are substantially homologous can be identifiedby comparing the sequences using standard software available in sequencedata banks, or in a Southern hybridization experiment under, forexample, stringent conditions as defined for that particular system.Defining appropriate hybridization conditions is within the skill of theart. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I & II,supra; Nucleic Acid Hybridization, supra. In particular, the heavy chainand light chain variable region sequences of the antibodies of thepresent invention are substantially homologous to a correspondinggermline gene sequence, having at least about 90% homology to acorresponding germline gene sequence.

It should be appreciated that also within the scope of the presentinvention are DNA sequences encoding an antibody of the invention, or apeptide analog, hapten, or active fragment thereof; which code for apeptide that defines in at least a portion thereof, or has the sameamino acid sequence as set forth in FIGS. 35-38, 45, 46, 55-61 and 71-72(SEQ ID NOS: 7, 8, 10, 9, 11, 12, 13, 14, 15, 16, 17, 18, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 49 and 51), but which aredegenerate to the same SEQ ID NOS. By “degenerate to” is meant that adifferent three-letter codon is used to specify a particular amino acid.It is well known in the art that the following codons can be usedinterchangeably to code for each specific amino acid:

Phenylalanine (Phe or F) UUU or UUC Leucine (Leu or L) UUA or UUG or CUUor CUC or CUA or CUG Isoleucine (Ile or I) AUU or AUC or AUA Methionine(Met or M) AUG Valine (Val or V) GUU or GUC of GUA or GUG Serine (Ser orS) UCU or UCC or UCA or UCG or AGU or AGC Proline (Pro or P) CCU or CCCor CCA or CCG Threonine (Thr or T) ACU or ACC or ACA or ACG Alanine (Alaor A) GCU or GCG or GCA or GCG Tyrosine (Tyr or Y) UAU or UAC Histidine(His or H) CAU or CAC Glutamine (Gln or Q) CAA or CAG Asparagine (Asn orN) AAU or AAC Lysine (Lys or K) AAA or AAG Aspartic Acid (Asp or D) GAUor GAC Glutamic Acid (Glu or E) GAA or GAG Cysteine (Cys or C) UGU orUGC Arginine (Arg or R) CGU or CGC or CGA or CGG or AGA or AGG Glycine(Gly or G) GGU or GGC or GGA or GGG Tryptophan (Trp or W) UGGTermination codon UAA (ochre) or UAG (amber) or UGA (opal)

It should be understood that the codons specified above are for RNAsequences. The corresponding codons for DNA have a T substituted for U.

Mutations can be made in a particular DNA sequence or molecule such thata particular codon is changed to a codon which codes for a differentamino acid. Such a mutation is generally made by making the fewestnucleotide changes possible. A substitution mutation of this sort can bemade to change an amino acid in the resulting protein in anon-conservative manner (i.e., by changing the codon from an amino acidbelonging to a grouping of amino acids having a particular size orcharacteristic to an amino acid belonging to another grouping) or in aconservative manner (i.e., by changing the codon from an amino acidbelonging to a grouping of amino acids having a particular size orcharacteristic to an amino acid belonging to the same grouping). Such aconservative change generally leads to less change in the structure andfunction of the resulting protein. A non-conservative change is morelikely to alter the structure, activity or function of the resultingprotein. The present invention should be considered to include sequencescontaining conservative changes which do not significantly alter theactivity or binding characteristics of the resulting protein.

The following is one example of various groupings of amino acids:

-   Amino Acids with Nonpolar R Groups-   Alanine-   Valine-   Leucine-   Isoleucine-   Proline-   Phenylalanine-   Tryptophan-   Methionine-   Amino acids with uncharged polar R groups-   Glycine-   Serine-   Threonine-   Cysteine-   Tyrosine-   Asparagine-   Glutamine

Amino Acids with Charged Polar R Groups (Negatively Charged at pH 6.0)

-   Aspartic acid-   Glutamic acid

Basic Amino Acids (Positively Charged at pH 6.0)

-   Lysine-   Arginine-   Histidine (at pH 6.0)

Another Grouping May be Those Amino Acids with Phenyl Groups:

-   Phenylalanine-   Tryptophan-   Tyrosine

Another grouping may be according to molecular weight (i.e., size of Rgroups):

Glycine 75 Alanine 89 Serine 105 Proline 115 Valine 117 Threonine 119Cysteine 121 Leucine 131 Isoleucine 131 Asparagine 132 Aspartic acid 133Glutamine 146 Lysine 146 Glutamic acid 147 Methionine 149 Histidine (atpH 6.0) 155 Phenylalanine 165 Arginine 174 Tyrosine 181 Tryptophan 204

Particularly preferred substitutions are:

-   -   Lys for Arg and vice versa such that a positive charge may be        maintained;    -   Glu for Asp and vice versa such that a negative charge may be        maintained;    -   Ser for Thr such that a free —OH can be maintained; and    -   Gln for Asn such that a free NH₂ can be maintained.

Amino acid substitutions may also be introduced to substitute an aminoacid with a particularly preferable property. For example, a Cys may beintroduced a potential site for disulfide bridges with another Cys. AHis may be introduced as a particularly “catalytic” site (i.e., His canact as an acid or base and is the most common amino acid in biochemicalcatalysis). Pro may be introduced because of its particularly planarstructure, which induces β-turns in the protein's structure.

Two amino acid sequences are “substantially homologous” when at leastabout 70% of the amino acid residues (preferably at least about 80%, andmost preferably at least about 90 or 95%) are identical, or representconservative substitutions. In particular, the heavy chain and lightchain variable region sequences of the antibodies of the presentinvention are substantially homologous to a corresponding germline geneamino acid sequence, having at least about 90%, and preferably at leastabout 95% homology to a corresponding germline gene amino acid sequence.

A “heterologous” region of the DNA construct is an identifiable segmentof DNA within a larger DNA molecule that is not found in associationwith the larger molecule in nature. Thus, when the heterologous regionencodes a mammalian gene, the gene will usually be flanked by DNA thatdoes not flank the mammalian genomic DNA in the genome of the sourceorganism. Another example of a heterologous coding sequence is aconstruct where the coding sequence itself is not found in nature (e.g.,a cDNA where the genomic coding sequence contains introns, or syntheticsequences having codons different than the native gene). Allelicvariations or naturally-occurring mutational events do not give rise toa heterologous region of DNA as defined herein.

As used herein, the term “antibody” is any immunoglobulin, includingantibodies and fragments thereof, that binds a specific epitope. Theterm is intended to encompass polyclonal, monoclonal, and chimericantibodies, the last mentioned described in further detail in U.S. Pat.Nos. 4,816,397 and 4,816,567. Such antibodies include both polyclonaland monoclonal antibodies prepared by known generic techniques, as wellas bi-specific or chimeric antibodies, and antibodies including otherfunctionalities suiting them for additional diagnostic use conjunctivewith their capability of modulating activity, e.g. that stimulates theremyelenation and/or regeneration of CNS axons, or that providesneuroprotection. An “antibody combining site” is that structural portionof an antibody molecule comprised of heavy and light chain variable andhypervariable regions that specifically binds antigen. The phrase“antibody molecule” in its various grammatical forms as used hereincontemplates both an intact immunoglobulin molecule and animmunologically active portion of an immunoglobulin molecule. Exemplaryantibody molecules are intact immunoglobulin molecules, substantiallyintact immunoglobulin molecules and those portions of an immunoglobulinmolecule that contains the paratope, including those portions know inthe art as Fab, Fab′, F(ab′)₂ and F(v).

Fab and F(ab′)₂ portions of antibody molecules, or antibody fragments,may be prepared by the proteolytic reaction of papain and pepsin,respectively, on substantially intact antibody molecules by methods thatare well-known. See for example, U.S. Pat. No. 4,342,566 toTheofilopolous et al. Fab′ antibody molecule portions are alsowell-known and are produced from F(ab′)₂ portions followed by reductionof the disulfide bonds linking the two heavy chains portions as withmercaptoethanol, and followed by alkylation of the resulting proteinmercaptan with a reagent such as iodoacetamide. An antibody containingintact antibody molecules is preferred herein.

The phrase “monoclonal antibody” in its various grammatical forms refersto an antibody having only one species of antibody combining sitecapable of immunoreacting with a particular antigen. A monoclonalantibody thus typically displays a single binding affinity for anyantigen with which it immunoreacts. A monoclonal antibody may thereforecontain an antibody molecule having a plurality of antibody combiningsites, each immunospecific for a different antigen; e.g., a bi-specific(chimeric) monoclonal antibody.

The general methodology for making monoclonal antibodies by hybridomasis well known. Immortal, antibody-producing cell lines can also becreated by techniques other than fusion, such as direct transformationof B lymphocytes with oncogenic DNA, or transfection with Epstein-Barrvirus. See, e.g., M. Schreier et al., “Hybridoma Techniques” (1980);Hammerling et al., “Monoclonal Antibodies And T-cell Hybridomas” (1981);Kennett et al., “Monoclonal Antibodies” (1980); see also U.S. Pat. Nos.4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,451,570; 4,466,917;4,472,500; 4,491,632; 4,493,890.

Panels of monoclonal antibodies useful in the present invention methodsor produced against neuromodulatory agent peptides or autoantibodypeptides can be screened for various properties; i.e., isotype, epitope,affinity, etc. Of particular interest are monoclonal antibodies thatexhibit the same activity as the neuromodulatory agents, andparticularly the present autoantibodies. Such monoclonals can be readilyidentified in activity assays such as the Theilers virus, EAE andlysolecithin models presented and illustrated herein. High affinityantibodies are also useful when immunoaffinity purification of native orrecombinant autoantibodies is possible.

Preferably, the antibody used in the diagnostic methods and therapeuticmethods of this invention is an affinity purified polyclonal antibody.More preferably, the antibody is a monoclonal antibody (mAb). Inaddition, it is contemplated for the antibody molecules used herein bein the form of Fab, Fab′, F(ab′)₂ or F(v) portions of whole antibodymolecules.

As suggested earlier, the diagnostic method of the present inventioncomprises examining a cellular sample or medium by means of an assayincluding an effective amount of an antagonist to an antibodypeptide/protein, such as an anti-peptide antibody, preferably anaffinity-purified polyclonal antibody, and more preferably a mAb. Inaddition, it is preferable for the anti-peptide antibody molecules usedherein be in the form of Fab, Fab′, F(ab′)₂ or F(v) portions or wholeantibody molecules. As previously discussed, patients capable ofbenefiting from this method include those suffering from a neurologicalcondition such as multiple sclerosis, Alzheimers disease, Parkinsonsdisease, a viral infection or other like neuropathological derangement,including damage resulting from physical trauma. Methods for isolatingthe peptides and inducing anti-peptide antibodies and for determiningand optimizing the ability of anti-peptide antibodies to assist in theexamination of the target cells are all well-known in the art.

Methods for producing polyclonal anti-polypeptide antibodies arewell-known in the art. See U.S. Pat. No. 4,493,795 to Nestor et al. Amonoclonal antibody, typically containing Fab and/or F(ab′)₂ portions ofuseful antibody molecules, can be prepared using the hybridomatechnology described in Antibodies—A Laboratory Manual, Harlow and Lane,eds., Cold Spring Harbor Laboratory, New York (1988), which isincorporated herein by reference. Briefly, to form the hybridoma fromwhich the monoclonal antibody composition is produced, a myeloma orother self-perpetuating cell line is fused with lymphocytes obtainedfrom the spleen of a mammal hyperimmunized with an antibodypeptide-binding portion thereof, or the antibody peptide or fragment, oran origin-specific DNA-binding portion thereof.

Splenocytes are typically fused with myeloma cells using polyethyleneglycol (PEG) 6000. Fused hybrids are selected by their sensitivity toHAT. Hybridomas producing a monoclonal antibody useful in practicingthis invention are identified by their ability to immunoreact in thesame fashion as the present autoantibodies and their ability to inhibitor promote specified activity in target cells and tissues.

A monoclonal antibody useful in practicing the present invention can beproduced by initiating a monoclonal hybridoma culture comprising anutrient medium containing a hybridoma that secretes antibody moleculesof the appropriate antigen specificity. The culture is maintained underconditions and for a time period sufficient for the hybridoma to secretethe antibody molecules into the medium. The antibody-containing mediumis then collected. The antibody molecules can then be further isolatedby well-known techniques.

Media useful for the preparation of these compositions are bothwell-known in the art and commercially available and include syntheticculture media, inbred mice and the like. An exemplary synthetic mediumis Dulbecco's minimal essential medium (DMEM; Dulbecco et al., Virol.8:396 (1959)) supplemented with 4.5 gm/l glucose, 20 mm glutamine, and20% fetal calf serum. An exemplary inbred mouse strain is the Balb/c.

Methods for producing monoclonal anti-peptide antibodies are alsowell-known in the art. See Niman et al., Proc. Natl. Acad. Sci. USA,80:4949-4953 (1983). Typically, the present antibody peptides, or apeptide analog or fragment, is used either alone or conjugated to animmunogenic carrier, as the immunogen in the before described procedurefor producing anti-peptide monoclonal antibodies. The hybridomas arescreened for the ability to produce an antibody that immunoreacts withthe antibody peptide analog and thereby reacts similarly to theantibodies of the present invention.

In the production of antibodies, screening for the desired antibody canbe accomplished by techniques known in the art, e.g., radioimmunoassay,ELISA (enzyme-linked immunosorbant assay), “sandwich” immunoassays,immunoradiometric assays, gel diffusion precipitin reactions,immunodiffusion assays, in situ immunoassays (using colloidal gold,enzyme or radioisotope labels, for example), western blots,precipitation reactions, agglutination assays, hemagglutination assays),complement fixation assays, immunofluorescence assays, protein A assays,and immunoelectrophoresis assays, etc. In one embodiment, antibodybinding is detected by detecting a label on the primary antibody. Inanother embodiment, the primary antibody is detected by detectingbinding of a secondary antibody or reagent to the primary antibody. In afurther embodiment, the secondary antibody is labeled. Many means areknown in the art for detecting binding in an immunoassay and are withinthe scope of the present invention.

Antibodies can be labeled for detection in vitro, e.g., with labels suchas enzymes, fluorophores, chromophores, radioisotopes, dyes, colloidalgold, latex particles, and chemiluminescent agents. Alternatively, theantibodies can be labeled for detection in vivo, e.g., withradioisotopes (preferably technetium or iodine); magnetic resonanceshift reagents (such as gadolinium and manganese); or radio-opaquereagents.

The labels most commonly employed for these studies are radioactiveelements, enzymes, chemicals which fluoresce when exposed to ultravioletlight, and others. A number of fluorescent materials are known and canbe utilized as labels. These include, for example, fluorescein,rhodamine, auramine, Texas Red, AMCA blue and Lucifer Yellow. Aparticular detecting material is anti-rabbit antibody prepared in goatsand conjugated with fluorescein through an isothiocyanate. Thepolypeptide can also be labeled with a radioactive element or with anenzyme. The radioactive label can be detected by any of the currentlyavailable counting procedures. The preferred isotope may be selectedfrom 3H, ¹⁴C, ³²P, ³⁵S, ³⁶Cl, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁹⁰Y, ¹²⁵I, ¹³¹I,and ¹⁸⁶Re.

Enzyme labels are likewise useful, and can be detected by any of thepresently utilized colorimetric, spectrophotometric,fluorospectrophotometric, amperometric or gasometric techniques. Theenzyme is conjugated to the selected particle by reaction with bridgingmolecules such as carbodiimides, diisocyanates, glutaraldehyde and thelike. Many enzymes which can be used in these procedures are known andcan be utilized. The preferred are peroxidase, β-glucuronidase,β-D-glucosidase, β-D-galactosidase, urease, glucose oxidase plusperoxidase and alkaline phosphatase. U.S. Pat. Nos. 3,654,090;3,850,752; and 4,016,043 are referred to by way of example for theirdisclosure of alternate labeling material and methods.

Polyclonal immunoglobulin preparations have been shown to exert abeneficial clinical effect in various clinical situations that arecharacterized or accompanied by a dysfunction or dysregulation of theimmune system. Immunoglobulin is also used to prevent or treat someillnesses that can occur when an individual does not produce enough ofits own immunity to prevent these illnesses. Nearly all immunoglobulinpreparations in use today are comprised of highly purified IgG, derivedfrom large pools of human plasma by fractionation. These preparationsare commonly administered intravenously (IVIG), although intramuscularadministration (IGIM) and oral administration is also used.

Commonly used IgG preparations include Gamimune (5% and 10%) (BayerCorporation), Gammagard (Baxter Healthcare Corporation), Polygam(American Red Cross), Sandoglobin (Sandoz Pharmaceuticals), Venoglobin(Alpha Therapeutic) and Intraglobin (Biotest Pharma GmbH). Anintramuscular immunoglobulin (IGIM), BayGam, is available from BayerCorporation. IVIG preparations in clinical use contain predominantlyIgG, smaller amounts of IgA, and yet smaller amounts of IgM, IgE andIgD, and generally comprise 95% or greater IgG, 2-5% IgA and traceamounts of IgM.

Pentaglobin (Biotest Pharma GmbH) is an IgM-enriched polyvalentimmunoglobulin preparation and each ml of solution comprises: IgM 6 mg;IgA 6 mg; IgG 38 mg and glucose monohydrate for injection 27.5 mg; or12% IgM, generally 10-15% IgM. Immunoglobulin preparations which havebeen further enriched for IgM can be readily generated and have beenreported as effective in animal models for treatment or alleviation ofcertain conditions. Riebert et al. report the use of IgM enriched humanintravenous immunoglobulin in a rat model of acute inflammation,particularly use of Pentaglobin and a laboratory preparation of IVIgM(35 g/l IgM, 12 g/l IgA, 3 g/l IgG). (Riebert, R. et al (1999) Blood93(3):942-951). Hurez et al report use of an intravenous IgM preparationof greater than 90% in experimental autoimmune ueveitis (EAU) (Hurez, V.et al (1997) Blood 90(10):4004-4013). IgM antibody immunoglobulinpreparations of at least 20% by weight IgM are described in U.S. Pat.Nos. 5,256,771, 5,510,465 and 5,612,033, incorporated herein byreference in their entity. Intravenously administerable polyclonalimmunoglobulin preparations containing at least 50% by weight of IgM interms of the total content of immunoglobulin are described by Moller etal in U.S. Pat. No. 5,190,752, incorporated herein by reference in itsentirety.

Immunoglobulin preparations are generated by methods and processesgenerally well known to those of skill in the art. Immunoglobulins areprepared from blood of healthy volunteers, where the number of blooddonors is at least about 5 or 10; preferably at least about 100; morepreferably at least about 1,000; still more preferably at least about10,000. In one common method, human plasma derived from pools ofthousands of donors is fractionated by cold ethanol fractionation (theCohn process or Cohn-Oncley process) (Cohn, et al (1946) J. Am. Chem.Soc. 68:459-475; Oncley, et al (1949) J. Am. Chem. Soc. 71:541-550)followed by enzymatic treatment at low pH, fractionation andchromatography. Cold ethanol fractionation may also be followed byultrafiltration and ion exchange chromatography. Further steps areincorporated to render immunoglobulin preparations safe from viraltransmission, including but not limited to enzymatic modification,chemical modification, treatment with beta-propiolactone, treatment atlow pH, treatment at high heat and treatment with solvent/detergent.Treatment with an organic solvent/detergent (S/D) mixture eliminatesviral transmission by enveloped viruses (HIV, hepatitis B, hepatitis C)(Gao, F. et al (1993) Vox Sang 64(4):204-9; U.S. Pat. Nos. 4,481,189 and4,540,573, incorporated herein by reference). Particular processes andmethods for preparation of IgM enriched immunoglobulin solutions aredescribed in U.S. Pat. Nos. 4,318,902 and 6, 136,132, which areincorporated herein by reference in their entirety.

Polyclonal IgM-enriched immunoglobulin preparations contemplated hereinand suitable for use in the methods of the present invention can be madeby any of the well-known methods used for preparing immunoglobulinpreparations. Suitable immunoglobulin preparations can also be obtainedcommercially. The immunoglobulin preparation can be a humanimmunoglobulin preparation. Suitable immunoglobulin preparations includeat least about 10% IgM, at least about 15% IgM, at least about 20% IgM,at least about 25% IgM, at least about 30% IgM, at least about 40% IgM,at least about 50% IgM, at least about 60% IgM, at least about 70% IgM,at least about 80% IgM, at least about 90% IgM and at least about 95%IgM. Polyclonal IgM immunoglobulin preparations suitable for use in thepresent invention include greater 10% IgM, greater than 20% IgM, andgreater than 50% IgM. Polyclonal IgM immunoglobulin preparationssuitable for use in the present invention include an amount of IgM whichis greater than the amount if IgG and greater than the amount of IgA.

Preparations of fragments of IgM enriched immunoglobulins, particularlyhuman immunoglobulins can also be used in accordance with the presentinvention. Fragments of immunoglobulins refer to portions of intactimmunoglobulins such as Fc, Fab, Fab′, F(ab)′₂ and single chainimmunoglobulins or monomers.

The IgM-enriched immunoglobulin preparation in preferably provided in apharmaceutically acceptable carrier, vehicle or diluent and isadministered intravenously, intramuscularly or orally. IgMimmunoglobulin is administered in doses and amounts similar to theadministration recognized and utilized by the skilled artisan for theadministration of clinically adopted immunoglobulins, including IVIG orIGIM or Pentaglobin, or as instructed or advised clinically or by themanufacturer. IgM preparations for use in the present invention areadministered in doses of about 0.5 mg/kg to about 1-2 g/kg body weightand can be administered as a single dose or in multiple separated dosesdaily or over the course of days or months. Suitable dosages include 10mg/kg body weight, 20 mg/kg body weight, 30 mg/kg body weight, 40 mg/kgbody weight, 50 mg/kg body weight, 75 mg/kg body weight, 100 mg/kg bodyweight, 200 mg/kg body weight, 300 mg/kg body weight, 400 mg/kg bodyweight, 500 mg/kg body weight, 1 g/kg body weight, and 2 g/kg bodyweight The polyclonal IgM immunoglobulin preparations may beadministered alone or in combination with other treatments, includingbut not limited to other compounds or agents for treatment oralleviation of the condition. In the instance of treatment oralleviation of a demyelinating disease, multiple sclerosis inparticular, the IgM immunoglobulin may be administered withanti-inflammatories, steroids, Betaseron, Copaxone, etc.

Accordingly, in one aspect of the diagnostic application of the presentinvention, a method is disclosed for detecting the presence or activityof a neuromodulatory agent, the neuromodulatory agent comprising amaterial selected from the group consisting of an antibody, a peptideanalog, a hapten, monomers thereof, active fragments thereof, agoniststhereof, mimics thereof, and combinations thereof, said neuromodulatoryagent having one or more of the following characteristics: inducingremyelination; binding to neural tissue; promoting Ca⁺⁺ signaling witholigodendrocytes; and, optionally, promoting cellular proliferation ofglial cells; wherein said neuromodulatory agent is measured by:

-   -   A) contacting a biological sample from a mammal in which the        presence or activity of said neuromodulatory agent is suspected        with a binding partner of said neuromodulatory agent under        conditions that allow binding of said neuromodulatory agent to        said binding partner to occur; and    -   B) detecting whether binding has occurred between said        neuromodulatory agent from said sample and the binding partner;    -   wherein the detection of binding indicates that presence or        activity of the neuromodulatory agent in the sample.

In a variant aspect, the invention extends to a method for detecting thepresence and activity of a polypeptide ligand associated with a giveninvasive stimulus in mammals comprising detecting the presence oractivity of the neuromodulatory agent as set forth above, wheredetection of the presence or activity of the neuromodulatory agentindicates the presence and activity of a polypeptide ligand associatedwith a given invasive stimulus in mammals. In a particular aspect, theinvasive stimulus is an infection, and may be selected from viralinfection, protozoan infection, bacterial infection, tumorous mammaliancells, and toxins.

In a further aspect, the invention extends to a method for detecting thebinding sites for a neuromodulatory agent, said neuromodulatory agentcomprising a material selected from the group consisting of an antibody,including antibodies of the IgM subtype and monomers thereof, a peptideanalog, a hapten, active fragments thereof, agonists thereof, mimicsthereof, and combinations thereof, said neuromodulatory agent having oneor more of the following characteristics: inducing remyelination;binding to neural tissue; promoting Ca⁺⁺ signaling witholigodendrocytes; and, optionally, promoting cellular proliferation ofglial cells; said method comprising:

-   -   A. placing a labeled neuromodulatory agent sample in contact        with a biological sample from a mammal in which binding sites        for said neuromodulatory agent are suspected;    -   B. examining said biological sample in binding studies for the        presence of said labeled neuromodulatory agent;    -   wherein the presence of said labeled neuromodulatory agent        indicates a binding site for a neuromodulatory agent.

Yet further, the invention includes a method of testing the ability of adrug or other entity to modulate the activity of a neuromodulatoryagent, said agent comprising a material selected from the groupconsisting of an antibody, including antibodies of the IgM subtype, apeptide analog, a hapten, monomers thereof, active fragments thereof,agonists thereof, mimics thereof, and combinations thereof, which methodcomprises:

-   -   A. culturing a colony of test cells which has a receptor for the        neuromodulatory agent in a growth medium containing the        neuromodulatory agent;    -   B. adding the drug under test; and    -   C. measuring the reactivity of said neuromodulatory agent with        the receptor on said colony of test cells;    -   wherein said neuromodulatory agent has one or more of the        following characteristics:        -   a) inducing remyelination;        -   b) binding to neural tissue, particularly oligodendrocytes;        -   b) promoting Ca⁺⁺ signaling with oligodendrocytes; and        -   c) promoting cellular proliferation of glial cells.

Correspondingly, the invention covers an assay method for screeningdrugs and other agents for ability to modulate the production or mimicthe activities of a neuromodulatory agent, said neuromodulatory agentcomprising a material selected from the group consisting of an antibody,a peptide analog, a hapten, monomers thereof, active fragments thereof,agonists thereof, mimics thereof, and combinations thereof, said methodcomprising:

-   -   A. culturing an observable cellular test colony inoculated with        a drug or agent;    -   B. harvesting a supernatant from said cellular test colony; and    -   C. examining said supernatant for the presence of said        neuromodulatory agent wherein an increase or a decrease in a        level of said neuromodulatory agent indicates the ability of a        drug to modulate the activity of said neuromodulatory agent,        said neuromodulatory agent having one or more of the following        characteristics:        -   i) inducing remyelination;        -   ii) binding to neural tissue, particularly oligodendrocytes;        -   iii) promoting Ca⁺⁺ signaling with oligodendrocytes; and        -   iv) promoting cellular proliferation of glial cells.

Lastly, a test kit is contemplated for the demonstration of aneuromodulatory agent in a eukaryotic cellular sample, saidneuromodulatory agent comprising a material selected from the groupconsisting of an antibody, including antibodies of the IgM subtype andmonomers thereof, a peptide analog, a hapten, active fragments thereof,agonists thereof, mimics thereof, and combinations thereof, which kitcomprises:

-   -   A. a predetermined amount of a detectably labeled specific        binding partner of a neuromodulatory agent, said neuromodulatory        agent having one or more of the following characteristics:        inducing remyelination; binding to neural tissue; promoting Ca⁺⁺        signaling with oligodendrocytes; and promoting cellular        proliferation of glial cells;    -   B. other reagents; and    -   C. directions for use of said kit.

A variant test kit is disclosed for demonstrating the presence of aneuromodulatory agent in a eukaryotic cellular sample, said agentcomprising a material selected from the group consisting of an antibody,a peptide analog, a hapten, monomers thereof, active fragments thereof,agonists thereof, mimics thereof, and combinations thereof. The kitcomprises:

-   -   A. a predetermined amount of a neuromodulatory agent, said        neuromodulatory agent having one or more of the following        characteristics: inducing remyelination; binding to neural        tissue; promoting Ca⁺⁺ signaling with oligodendrocytes; and        promoting cellular proliferation of glial cells;    -   B. a predetermined amount of a specific binding partner of said        neuromodulatory agent;    -   C. other reagents; and    -   D. directions for use of said kit;    -   wherein either said neuromodulatory agent or said specific        binding partner are detectably labeled. Both of the above kits        may utilize a labeled immunochemically reactive component        selected from the group consisting of polyclonal antibodies to        the neuromodulatory agent, monoclonal antibodies to the        neuromodulatory agent, fragments thereof, and mixtures thereof.

The present invention extends to the use and application of theantibodies of the present invention, particularly autoantibodies,including antibodies of the IgM subtype and monomers thereof, ormixtures and/or active fragments thereof, characterized by their abilityto bind to structures and cells in the central nervous system,particularly including oligodendrocytes, in imaging and in vivodiagnostic applications. Thus, the antibodies, by virtue of theirability to bind to structures and cells in the central nervous system,particularly including oligodendrocytes, can be utilized viaimmunofluorescent, radioactive and other diagnostically suitable tags asimaging agents or imaging molecules for the characterization of thenervous system, including the central nervous system and the diagnosis,monitoring and assessment of nervous disease, particularly includingmultiple sclerosis. The antibodies may further be utilized as imagingagents or imaging molecules in the diagnosis, monitoring and assessmentof stroke, spinal cord injury, and various dementias includingAlzheimer's disease. The appropriate and suitable immunofluorescent,radioactive, or other tagging molecules or agents for coupling orattachment to the antibodies for use in in vivo imaging will be wellknown to and within the skill of the skilled artisan.

The present invention also relates to methods of treating demyelinatingdiseases in mammals, such as multiple sclerosis in humans, and viraldiseases of the central nervous system of humans and domestic animals,such as post-infectious encephalomyelitis, using the SCH 94.03, SCH79.08, O1, O4, A2B5 and HNK-1 monoclonal antibodies, and the humanautoantibodies sHIgM22, ebvHIgM MSI19D10, sHIgM46, analogs thereofincluding haptens, active fragments thereof, or a natural or syntheticautoantibody having the characteristics thereof. Methods of prophylactictreatment using these mAb, active fragments thereof, or other natural orsynthetic autoantibodies having the same characteristics, to inhibit theinitiation or progression demyelinating diseases are also encompassed bythis invention.

Oligodendrocytes (OLs), the myelin-forming cells of the central nervoussystem (CNS), originate as neuroectodermal cells of the subventricularzones, and then migrate and mature to produce myelin. The sequentialdevelopment of OLs is identified by well-characterized differentiationstage-specific markers. Proliferative and migratory bipolar precursors,designated oligodendrocyte/type-3 astrocyte (0-2A) progenitors, areidentified by monoclonal antibodies (mAbs) anti-GD₃ and A2B5 [Eisenbarthet al., Proc. Natl. Acad. Sci. USA, 76 (1979), 4913-4917]. The nextdevelopmental stage, characterized by multipolar, postmigratory, andproliferative cells, is recognized by mAb O4 [Gard et al., Neuron, 5(1990), 615-625; Sommer et al., Dev. Biol., 83 (1981), 311-327]. Furtherdevelopment is defined by the cell surface expression ofgalactocerebroside, recognized by mAb 01 [Schachner, J. Neurochem., 39(1982), 1-8; Sommer et al., supra], and by the expression of2′,3′-cyclic nucleotide 3′-phosphohydrolase. The most mature cellsexpress terminal differentiation markers such as myelin basic proteinand proteolipid protein.

The mAbs (A2B5, O1, and O4) used to characterize the stages of OLdevelopment were made by immunizing BALB/c mice with chicken embryoretina cells or homogenate of bovine corpus callosum [Eisenbarth et al.,supra; Sommer et al., supra]. A2B5 recognizes not only O-2A progenitorsbut also neurons and reacts with cell surface ganglioside GQ1c [Kasai etal., Brain Res., 277 (1983), 155-158] and other gangliosides [Fredman etal., Arch. Biochem. Biophys., 233 (1984), 661-666]. 04 reacts withsulfatide, seminolipid and cholesterol [Bansal et al., J. Neurosci.Res., 24 (1989), 548-557], whereas O1 reacts with galactocerebroside,monogalactosyl-diglyceride and psychosine [Bansal et al., supra]. ThesemAbs belong to the IgM immunoglobulin (Ig) subclass and recognizecytoplasmic structures as well as the surface antigens of OLs[Eisenbarth et al., supra; Sommer et al., supra]. Mouse mAb HNK-1(anti-Leu-7), made by immunizing BALB/c mice with the membranesuspension of HSB-2 T lymphoblastoid cells, was first reported as amarker for natural killer cells [Abo et al., J. Immunol., 127 (1981),1024-1029]. Later, HNK-1 was shown to share antigenic determinants withthe nervous system [Schuller-Petrovic et al., Nature, 306 (1983),179-181]. The carbohydrate epitope on myelin-associated glycoprotein,found in both central and peripheral myelin sheaths, was shown to be aprincipal antigen of nervous tissue the reacted with HNK-1 [McGarry etal., Nature, 306 (1983), 376-378]. However, other glycoproteins innervous tissue react with this mAb, some of which are important inembryogenesis, differentiation, and myelination [Keilhauer et al.,Nature, 316 (1985), 728-730; Kruse et al., Nature, 311 (1984), 153-155;Kruse et al., Nature, 316 (1985), 146-148; McGarry et al., J.Neuroimmunol., 10 (1985), 101-114]. Of interest, HNK-1 also reacts withcytoplasmic structures and belongs to the IgM Ig subclass.

A monoclonal antibody, disclosed and claimed in copending parentapplication U.S. Ser. No. 08/236,520, filed Apr. 29, 1994, anddesignated SCH94.03, was found to promotes CNS remyelination in miceinfected chronically with Theiler's murine encephalomyelitis virus(TMEV) [Miller et al., J. Neurosci., 14 (1994), 6230-6238]. SCH94.03belongs to the IgM(χ) Ig subclass and recognizes an unknown surfaceantigen on OLs, but cytoplasmic antigens in all cells (Asakura et al.,Molecular Brain Research, in press). The polyreactivity of SCH94.03 byELISA, and the unmutated Ig variable region germline sequences indicatedthat SCH94.03 is a natural autoantibody [Miller et al., J. Neurosci., 14(1994), 6230-6238]. A close study of SCH94.03, and comparison thereofwith well-known OL-reactive mAbs A2B5, O1, O4, and HNK-1 raised thepossibility that these are natural autoantibodies. A subsequent analysisof the Ig variable region cDNA sequences and the polyreactivity of thesemAbs by ELISA confirmed that this is a generic group of naturalautoantibodies having similar utilities.

The antigen reactivity of the monoclonal antibody, IgM monoclonalantibody referred to herein as SCH 94.03 (also referred to herein asSCH94.32) and SCH 79.08 (both prepared from a mammal immunized withspinal cord homogenate from a normal mammal (i.e., uninfected with anydemyelinating disease)), have been characterized and described in theaforesaid parent Application U.S. Ser. No. 08/236,520, filed Apr. 29,1994, whose teachings are incorporated herein by reference, usingseveral biochemical and molecular assays, includingimmunohistochemistry, immunocytochemistry, Western blotting, solid-phaseenzyme-linked immunosorbant assays (ELISA), and Ig variable regionsequencing. The hybridomas producing monoclonal antibody SCH 94.03 andSCH 79.08 have been deposited on Apr. 28, 1994, and Feb. 27, 1996,respectively, under the terms of the Budapest Treaty, with the AmericanType Culture Collection (ATCC) and have been given ATCC Accession Nos.CRL 11627 and HB12057, respectively. All restrictions upon theavailability of the deposit material will be irrevocably removed upongranting of a patent.

Natural or physiologic autoantibodies are present normally in serum, arecharacterized by being reactive or capable of binding to selfstructures, antigens or cells. They are often polyreactive, arefrequently of the IgM subtype, and are encoded by unmutated germlinegenes or are substantially homologous to germline genes with few orseveral sequence differences. By sequencing immunoglobulin (Ig) cDNAs ofthe oligodendrocyte-reactive O1, O4, A2B5, and HNK-1 IgM χ monoclonalantibodies and comparing these with published germline sequences, it wasdetermined that these were natural autoantibodies. O1 V_(H) wasidentical with unrearranged V_(H) segment transcript A1 and A4, O4 V_(H)had three and HNK-1 V_(H) had six nucleotide differences from V_(H)101in the V_(H) coding region. The D segment of O1 was derived fromgermline SP2 gene family, J_(H)4, whereas O1 J_(H) was encoded bygermline J_(H)1 with one silent nucleotide change. O1 and O4 lightchains were identical with myeloma MOPC21 except for one silentnucleotide change.

HNK-1 V_(χ) was identical with germline V_(χ)41 except for two silentnucleotide changes. O1 J_(χ), O4J_(χ) and HNK J_(χ) were encoded byunmutated germline J_(χ)2. In contrast, A2B5 V_(H) showed sevennucleotide differences from germline V1, whereas no germline sequenceencoding A2B5 V_(χ) was identified. O1 and O4, but not A2B5 werepolyreactive against multiple antigens by direct ELISA. Therefore, O1,O4 and HNK-1 Igs are encoded by germline genes, and have the genotypeand phenotype of natural autoantibodies.

Selection of SCH mAbs to Promote CNS Remyelination

A panel of monoclonal antibodies (mAbs) derived from splenocytes ofuninfected SJL/J mice injected with SCH was constructed as described indetail in Example 1. After the initial fusion and cloning, 2 of the 95wells with viable Ig-secreting hybridomas contained mAb with significantbinding to SCH as demonstrated by ELISA. Hybridoma cells from these twowells, called the 79 and 94 series, were subcloned by limiting dilutionand screened again for binding to SCH by ELISA. For the 79 serieshybridomas, 14 out of 49 clones were positive by SCH ELISA, while forthe 94 series, 17 out of 32 were positive for binding to SCH. Based uponthe ELISA data, two 79 series hybridomas (SCH79.08 and SCH79.27), bothof which also reacted with myelin basic protein (MBP) by ELISA, andthree 94 series hybridomas (SCH94.03, SCH94.11, and SCH94.32), none ofwhich reacted with MBP, were chosen for ascites production and in vivotransfer experiments.

mAbs Promote Proliferation of Glial Cells

As described in Example 2, the mAbs were tested for their ability topromote proliferation of glial cells in vitro.

The dose-response characteristic of antibody-mediated proliferation werethen examined. As shown in FIG. 1, maximal stimulation with 94.03 wasseen at 100 ng/ml. Control myeloma IgMs MOPC 104E and TEPC 183 (data notshown) also stimulated the mixed rat brain cultures to proliferate.However, the maximal effect was seen at a 10-fold higher concentrationthan that seen with the mAbs.

The temporal profile of antibody-mediated proliferation was alsoexamined as shown in FIG. 2. On day 8, after culture initiation, 100ng/ml antibody was added to the cultures (time 0). Cells were harvestedat 24 hour intervals; [³H]thymidine was present for the final 24 hoursof culture to measure the total proliferation during the interval. Themaximal stimulation with 94.03 was seen at 72 hours after antibodyaddition. Similar results were obtained with 94.32. None of the isotypecontrol antibodies showed any significant proliferation throughout the120 hours of culture. These data demonstrates that both mAbs 94.32 and94.03 induce proliferation of glial cells of mixed rat brain culture.This proliferation is maximal at an antibody concentration of 100 ng/mland a culture period of 72 hours after antibody addition.

CNS Remyelination Promoted by mAbs SCH94.033 and SCH94.32

As described in Example 3, SJL/J mice chronically infected with TMEVwere treated with a total mAb dose of 0.5 mg iv or 5.0 mg ip dividedinto twice weekly doses for 4-5 weeks. CNS remyelination was measured bya quantitative morphological assessment on ten spinal cordcross-sections from each mouse. The criterion for CNS remyelination wasabnormally thin myelin sheaths relative to axonal diameter. The data arecomposite of six experiments and are presented as the ±SEM, where nindicates the number of mice. Statistical comparisons for remyelinationdata were made with the cumulative values from both IgM and buffer onlycontrols using a modified rank sum test. The number of demyelinatedlesions and the area of demyelination were not significantly differentbetween treatment groups assessed by a one-way ANOVA. For control IgMs,myelomas MOPC 104E and ABPC 22 (both from Sigma), and TB5-1, ananti-mycobacteria mAb, were used.

SJL/J mice chronically infected with TMEV and treated with either mAbSCH94.03 or SCH94.32 showed significantly greater CNS remyelination thananimals treated with either isotype-matched control mAb or buffer onl+(Table 1).

TABLE 1 Monoclonal antibodies SCH94.03 and SCH94.32 promote CNSremyelination Area Number of Number of Area of Remyelination/Demylination Remyelinated Remyelination Area of Lesion area LesionTreatment n (lesions) Lesions p-value (mm²) (mm²) (%) SCH94.03 12 25.6 ±2.6 12.8 ± 2.6 <0.0025 0.35 ± 0.09 1.09 ± 0.19 28.9 ± 3.8 SCH94.32 1224.9 P ± 2.8  12.3 ± 2.3 <0.0001 0.42 ± 0.11 1.46 ± 0.21 26.7 ± 4.2 IgMcontrol 13 29.9 ± 2.0  6.7 ± 1.2 — 0.11 ± 0.02 1.70 ± 0.28  7.7 ± 1.8Buffer only 11 27.7 ± 2.7  5.1 ± 1.1 — 0.06 ± 0.01 1.11 ± 0.29  6.5 ±1.2

Remyelination was seen with either iv or ip injections. SCH94.03- orSCH94.32-treated animals had approximately 2-3-fold more remyelinatedlesions, and a 3-4-fold larger total area of CNS remyelination thancontrol animals. When a cumulative statistical comparison was made usingthese two parameters of therapeutic effectiveness, the CNS remyelinationinduced by mAbs SCH94.03 and SCH94.32 was highly significant (p<0.005;Table 2). In a chronic progressive disease like TMEV infection, theextent of CNS repair is a direct function of the extent of CNS damage.Both the number and area of CNS lesions were not different betweentreatment groups, indicating similar disease severity (Table 1). WhenCNS remyelination was expressed as the percentage of lesion area showingremyelination, approximately one-third of the cumulative demyelinatedlesion area shown CNS remyelination in mice treated with either mAbSCH94.03 or SCH94.32 (Table 1).

Similar results were obtained using Schh 79.08 (Results shown in Table2) and for O1, O4, A2B5 and HNK-1 (Results shown in Table 3).

TABLE 2 Enhancement of CNS remyelination by SCH79.08 Area of CNS-typeArea of CNS-type Area of remyelination/ Area of white remyelinationdemyelinated area of Treatment No. of Mice matter (mm²) (mm²) lesion(mm²) lesions (%) SCH79.08 15 8.42 ± 0.33 0.20 ± 0.05 1.01 ± 0.16 20.2 ±4.7 PBS 6 8.89 ± 0.26 0.03 ± 0.01 1.01 ± 0.21  2.4 ± 0.8 Valuesrepresent the mean ± SEM. Statistics by student t-test comparing area ofCNS-type remyelination/area of lesions revealed p < 0.05. PBS: phosphatebuffered saline.

TABLE 3 Enhancement of CNS remyelination by oligodendrocyte-reactivemonoclonal antibodies Area of CNS-type Area of CNS-type Area ofremyelination/ Area of White remyelination demyelinated area ofTreatment No. of mice matter (mm²) (mm²) lesion (mm²) lesions (%) O1 67.57 ± 0.52 0.14 ± 0.04 0.53 ± 0.10 24.8 ± 6.2* O4 7 8.01 ± 0.15 0.17 ±0.04 0.84 ± 0.10 20.4 ± 4.2* A2B5 7 7.28 ± 0.38 0.18 ± 0.05 0.70 ± 0.1724.6 ± 4.6* HNK-1 7 7.16 ± 0.38 0.15 ± 0.03 0.78 ± 0.10 20.6 ± 2.8* PBS6 7.46 ± 0.70 0.05 ± 0.02 0.51 ± 0.14 8.0 ± 2.2 Values represent themean ± SEM. Statistics by student t-test comparing area of CNS-typeremyelination/area of lesions revealed *p < 0.05, **p < 0.01. PBS:phosphate buffered saline

Morphology of CNS Remyelination

CNS remyelination was readily identified morphologically both by lightand electron microscopy (FIG. 3A-3D). FIG. 3A shows a remyelinatedlesion from an animal treated with SCH94.03. The majority of axons inthe lesion show morphologic evidence of repair, with abnormally thinmyelin sheaths relative to axonal diameter (Ludwin, S. K. “Remyelinationin the central nervous system of the mouse,” In: THE PATHOLOGY OF THEMYELINATED AXON (Adachi M, Hirano A, Aronson S M eds), pp 49-79, Tokyo:Igaku-Shoin Ltd. (1985)). For comparison, FIG. 3B shows a demyelinatedlesion, with minimal remyelination, whereas FIG. 3C is an area of normalmyelin, with thickly myelinated axons. Within remyelinated lesions (FIG.3A), there were 15.3±1.0 (mean±SEM) myelinated axons per 100 μm²,compared to only 1.1±0.2 myelinated axons per 100 μm² in demyelinatedlesions (FIG. 3B). FIG. 3C shows a light micrograph of spinal cordsection with normal myelin. By electron microscopy, CNS remyelinationwas especially evident (FIG. 3D). Almost every axon in the field hasevidence of new myelin formation, although the degree of remyelination(i.e., myelin thickness) is variable between individual axons,suggesting different stages of the repair process. The ratio of myelinthickness to axonal diameter was 0.08±0.01 (mean±SEM; n=25 axons) forremyelinated axons compared to 0.21±0.01 (n=34 axons) for normallymyelinated axons.

Correlation Between Clinical Disease and Morphological Remyelination

The correlation of morphological remyelination with clinical signs ofdisease improvement was assessed as described in Example 3. At eachtreatment injection, mice were assessed clinically as described inExample 3. The change in clinical score was correlated with thepercentage of lesion area showing remyelination (FIG. 4). Morphologicalremyelination is represented as the percentage of lesion area showingCNS remyelination. A change in clinical score of 0 represent stabledisease over the treatment period (4-5 weeks), whereas a positive changeindicates worsening of clinical disease, and a negative change indicatesimprovement. Data represent individual animals from all treatmentgroups. A positive change in clinical score indicates worsening ofdisease. Using data from all treatment groups, the change in clinicalscore showed a moderate but significant negative correlation (R=−0.40;p<0.04) with the percentage of lesion area showing remyelination.Although few animals actually improved clinically (Δ clinical score <0),animals with an increase in disease severity (Δ clinical score >0)tended to have less morphological remyelination, while animals thatremained stable clinically (Δ clinical score=0) showed the mostremyelination. A similar negative correlation was obtained when theother quantitative measures of remyelination were used (the number ofremyelinated lesions and the area of remyelination) as shown in Table 1.These data demonstrate that remyelination quantitated by morphology isassociated with slowing of clinical disease progression.

Titration of mAb SCH94.03 Dose and CNS Remyelination

For the initial treatment experiments, a total mAb dose of 25 mg/kg forintravenous (iv) injections and 250 mg/kg for intraperitoneal (ip)injection was empirically chosen. To assess the dose-responsecharacteristics, and to determine the minimal amount of mAb needed topromote remyelination, chronically-infected mice were treated withvarious ip doses of SCH94.03. Remyelination was quantitated as describedfor Table 1. Data are the mean values of 4-5 animals per mAb dose, withthe final cumulative dose indicated on the graph. SEM averaged 35% ofthe mean. There was no statistical difference assessed by one-way ANOVAin the number of demyelinated lesions or the area of demyelinationbetween treatment groups, indicated similar extent of disease in allanimals. The number of demyelinated lesions and area of lesions were33.2±7.5 and 1.25±0.43 for the 1000 μg group, 31.8±8 and 1.11±0.31 forthe 100 μg group, 23.8±3.4 and 0.54±0.14 for the 10 μg group, and20.0±6.5 and 0.74±0.20 for the buffer only group (represented as the 0dose point on the graph). Animals treated with 100 μg control IgM (MOPC104E) had remyelination scores similar to control animals treated withbuffer only. The positive correlation between the dose of mAb SCH94.03and CNS remyelination was especially striking when the severity of CNSdisease was taken into account. When CNS repair was expressed as thepercentage of lesion area showing remyelination, mice treated with atotal dose of 1000, 100, or 10 μg of SCH94.03 had 6-, 5-, and 4-foldmore remyelination than control animals, respectively (FIG. 5). Micegiven as little as 10 μg of SCH94.03 ip (0.5 mg/kg) showed evidence ofenhanced CNS remyelination. These data indicated that mAb SCH94.03 andCNS remyelination had a positive does-response relationship, and thatvery small quantities of mAb were needed to promote myelin repair.

Antigen Specificity of SCH94.03 and SCH94.32

Although mAbs SCH94.03 and SCH94.32 were generated from splenocytes ofuninfected mice, and screened against SCH from uninfected mice, it wasdirectly assessed whether either mAb could react with TMEV capsidproteins or inhibit viral infectivity in vitro. By Western blotting(FIG. 6), SCH94.03 and SCH94.32 did not react with any TMEV proteinsrecognized by either serum from chronically infected mice or polyclonalIgG from rabbits injected with purified TMEV (Rodriguez, et al., Ann.Neurol., 13:426-433 (1983)). Western blot of lysates from control mockinfected L2 cells showed single bands with the serum from chronicallyinfected animals and the polyclonal rabbit anti-TMEV IgG at 32 and 43kDa, respectively, but no reactivity with SCH94.03 or SCH94.32.

In addition, no significant inhibition of TMEV infectivity in vitro withup to 5 μg/ml of either SCH94.03 or SCH94.32, was observed under assayconditions where 50% neutralization was observed with a 1:34,000dilution of serum from chronically infected animals. These resultsindicated that the therapeutic effect of SCH94.03 and SCH94.32 was notdue to direct inhibition of the virus.

To initially characterize the antigens recognized by mAbs SCH94.03 andSCH94.32, various cell lines derived from glial (rat C6, mouse G26-20,human U373MG and U87MG), neural (human neuroblastoma), fibroblast (mouseL and 3T3), epithelial (human SCC-9 carcinoma), and lymphocytic (mouseCTLL2) origin were stained. Both mAbs stained internal antigens of allcell lines tested, which indicated that certain antigens recognized bythese mAbs were not restricted to unique cell types in vitro. Based onthe hypothesis that the therapeutic effect of SCH94.03 and SCH94.32 wasdue to a CNS-specific interaction, the immunostaining of cultured cellsby SCH94.03 and SCH94.32 using the rat glial cell line 5.5B8 was furtherinvestigated. This immortalized glial cell line has phenotypiccharacteristics of both oligodendrocytes and astrocytes, with expressionof MBP and 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNP), and low,but detectable, expression of glial fibrillary acidic protein (GFAP) andthe lipids or proteins recognized by the mAbs A2B5 and 04 (Bozyczko, etal., Ann. NY Acad. Sci., 605:350-353 (1990)). SCH94.03 and SCH94.32recognized both a surface and cytoplasmic determinant on 5.5B8 cells.The surface staining was most prominent on small cells which lay on topof a layer of flat, morphologically differentiated cells (FIG. 7A).Surface staining was confirmed by flow cytometry on live cells. When thecell membrane was permeabilized by dehydration or brief treatment with anon-ionic detergent to expose internal antigens, the staining patternwas altered considerably (FIG. 7B). The cytoplasmic staining wasfilamentous, with a dense perinuclear network that extended out into thecell processes. This pattern closely resembled the staining pattern ofthe intermediate filament cytoskeletal protein vimentin. These dataindicated that SCH94.03 and SCH94.32 recognized antigens that were notrestricted to cells derived from the nervous system, but that they didrecognize both surface and cytoplasmic determinants on glial cells.

Immunohistochemical staining of frozen mouse, rat, and human tissueconfirmed that SCH94.03 and SCH94.32 were not CNS-specific mAbs, butrather showed multi-organ reactivity. Both mAbs immunostained all majororgans examined, including the brain, spinal cord, optic nerve, heart,liver, kidney, stomach, and small intestine and skeletal muscle.However, not all cells within an organ stained, suggesting in situcytological specificity. Within the CNS, SCH94.03 and SCH94.32 stainedpredominately blood vessels, ependymal cells, and stellate-shaped cellswith the morphological features of glial cells, which were enriched inneonatal cerebellar, periventricular, and brain stem white matter (FIG.7C), and both neonatal and adult optic nerve. Similar glial cellspositive for SCH94.03 and SCH94.32 were found in autopsied human braintissue, especially at the gray-white matter junction (FIG. 7D).Identical immunostaining results were obtained with mAb SCH94.32.Immunostaining with a control IgM (MOPC 104E) was negative for allsamples and tissue structures which immunostained with SCH94.03 andSCH94.32.

The identification and characterization of an entire family ofautoantibodies, referred to as “natural” or “physiological”autoantibodies, has influenced traditional view of autoimmunity andself-reactivity. The autoantibodies that have been studied extensivelyare typically IgMs, although other isotypes have been identified, arereactive toward a wide range of self structures or antigens, includingcytoskeletal proteins, surface proteins, nucleic acids, phospholipids,bacterial antigens such as lipopolysaccharides, and various chemicalhaptens (reviewed by Avrameas and Ternynck, Mol. Immunol., 30:1133-1142(1993)). Natural autoantibodies share extensive idiotypiccross-reactivity or “connectivity”, which includes expression of similaridiotypes, some of which are expressed by pathogenic autoantibodies, aswell as reactivity toward common idiotypes expressed on otherantibodies. Molecular analysis has shown that natural autoantibodies aretypically encoded by unmutated germline immunoglobulin (Ig) genes, orsubstantially homologous thereto, with few or several somatic mutations,and therefore represent a substantial fraction of the Ig repertoire,especially in neonatal animals which have not had extensive exogenousantigen exposure.

The function of natural autoantibodies remains enigmatic. Severalhypotheses have been proposed based upon their biochemical and molecularcharacteristics. These include: (1) clearance of senescent or damagetissue, (2) providing a first line of immunological defense in the lagperiod between pathogen exposure and an Ag-specific immune response, (3)masking autoantigens from a potentially pathogenic autoimmune response,(4) immunomodulation, including shaping of the neonatal immunerepertoire via an idiotypic network, and (5) participation in thepositive selection of B cells in the bone marrow, similar to the processproposed for T cells in the thymus.

The hypothesis that antibodies SCH94.03 and SCH94.32 were naturalautoantibodies was tested. To characterize the antigen reactivities ofSCH94.03 and SCH94.32, several biochemical and molecular assays,including immunohistochemistry and immunocytochemistry, Westernblotting, solid-phase enzyme-linked immunosorbant assays (ELISA), and Igvariable region sequencing, were used. As described below, for allbiochemical assays, SCH94.03 and SCH94.32 were indistinguishable. Inaddition, SCH94.03 and SCH94.32 had identical Ig variable regionsequences, which confirmed that they were the same mAb. Further detailsof these characterizing studies are reported in Asakura et al., J.Neuroscience Res. (1996) 43, pp 273-281, which disclosure isincorporated herein by reference.

A potential mechanism whereby SCH94.03 could stimulate remyelination inthe central nervous system would be to stimulate the proliferationand/or differentiation of cells involved in myelinogenesis, primarilyoligodendrocytes or their immature precursors. Thus, it was testedwhether SCH94.03 stained the surface of various cells. Usingimmortalized cells, it was determined that SCH94.03 stained two glialcells lines, 5.5B8 (FIG. 7A) and 20.2E11, but did not stain the surfaceof several other glial cells lines (10.1A3, 20.2A40, C6, G26-20), aneuroblastoma cell line (B104), two fibroblast lines (L2, Cos-1), or twomyoblastomas (G8, L6). Similar results were obtained with cells isolatedfrom animal tissues and grown in culture. SCH94.03 stained the surfaceof oligodendrocytes, but not astrocytes, microglia, Schwann cells,myoblasts, or fibroblasts.

The reactivity of SCH94.03 with proteins from glial and lymphoid celllines, and tissue lysates from brain, liver, and intestine by Westernblotting was also assessed. SCH94.03 reacted with multiple bands fromall cells and tissues examined, with prominent reactivity towards bandsat 50, 95, 120, and >200 kDa. The exact identity of these protein bandshas not been determined.

The activity of SCH94.03 with several purified protein self-antigens bysolid-phase ELISA was determined. (FIG. 8A-8C). SCH94.03 showed strongreactivity toward the RBC antigen spectrin, but also showed consistentreactivity toward hemoglobin, actin, tublin, and vimentin, andthyroglobulin, although to a lesser qualitative degree than towardspectrin. No reactivity was observed with myosin, transferrin, albumin,lysozyme, or myelin basic protein under our assay conditions. Six othermonoclonal or myeloma IgM controls XXMEN-0E5 (FIG. 8B), A2B5, MOPC104E,TEPC183, 01, and CH12 (FIG. 8C), were also tested, and no reactivitywith any of the antigens tested was observed.

To confirm the monoclonality of SCH94.03, 18 subclones of SCH94.03 (9each from SCH94.03 and SCH94.32 parents) were tested for polyreactivityby solid-phase ELISA. All 18 subclones showed identical reactivitypatterns with the panel of protein antigens as the parent SCH94.03. Tofurther support the conclusion that the polyreactivity of SCH94.03 wasvia its Fab region, we generated F(ab)₂′ fragments and assessed theirreactivity with the protein antigens by ELISA (FIG. 9). SCH94.03 F(ab)₂′fragments showed similar polyreactivity as the whole IgM molecule.

A panel of chemical haptens coupled to bovine serum albumin (BSA) wasconstructed and used to assess SCH94.03 reactivity by solid-phase ELISA(FIG. 10A-10C). SCH94.03 showed strong reactivity toward fluorescein(FL) and 4-hydroxy-3-nitrophenyl acetic acid (NP), moderate reactivitytoward phenyloxazolone (PhOx), and weak reactivity toward 2, 4,6-trinitrophenyl (TNP) and p-azophenylarsonic acid (Ars). No reactivitywith p-azophenyltrimethylammonium (TMA), p-azophenylphosphorylcholine(PC), or the carrier protein BSA was detected. Control IgMs (FIGS. 10Band 10C) showed no significant binding to any of the haptens tested,with the exceptions of CH12 reactivity with TMA, which has beenpreviously reported, and A2B5 reactivity with NP.

It was further investigated whether the Ig light (L) and heavy (H)chains of SCH94.03 were encoded by germline Ig genes (FIG. 11). Thelight chain variable (V_(L)) and joining (J_(L)) region nucleotidesequences from SCH94.03 had 99.4% identity with the previously publishedsequences of the germline Vκ10 and Jκ1 genes, with only two silentchanges at the 3′ end of both the V_(L) and J_(L) regions. The SCH94.03V_(H) region nucleotide sequence was identical to the previouslypublished germline V_(H)23 sequence, the J_(H) region sequence differedfrom the published germline J_(H)2 sequence by one nucleotide, at the 5′end of the J region, and the diversity (D) region contained 15contiguous nucleotides derived from the germline DFL16.1 gene. Therewere 8 nucleotides in the V-D junction, and 1 in the D-J junction, whichdid not correspond to any known germline V or D region genes, andprobably represent noncoded (N) nucleotides inserted by the enzymeterminal deoxynucleotide transferase during V-D-J recombination. Theonly changes from the germline genes in the heavy chain of SCH4.03occurred at either the V-D or D-J junction, and therefore couldrepresent either N nucleotides or the result of imprecise joining,rather than somatic mutations. In addition, both the light and heavychain variable regions of SCH94.03 showed extensive sequence similaritywith the IgM produced by the B-cell lymphoma CH12 (FIG. 11).

These antigen reactivity results suggest that SCH94.03 is a naturalautoantibody. Although this conclusion does not readily present amechanism as to how SCH94.03 stimulates remyelination in the centralnervous system, it does suggest an important physiological function ofnatural autoantibodies. Autoantibodies that are produced either duringnormal physiology, or in response to tissue damage and the subsequentrelease of previously sequestered antigens, might actively participateto promote repair in the damaged tissue. In line with previouslyproposed functions of natural autoantibodies, this active participationmight be to facilitate removal of damaged tissue, mask autoantigensthereby preventing vigorous pathogenic autoimmune response, modulate theimmune response which actually resulted in the tissue destruction,thereby allowing normal endogenous tissue repair to occur, or directlystimulate cells involved in the repair process.

Thus, as a result of the work described herein, it is now demonstratedthat an autoantibody generated and screened for its autoantigen-bindingcapability, also promotes CNS remyelination. Mice chronically infectedwith TMEV and treated either intravenously (iv) or intraperitoneally(ip) with IgM mAbs from hybridomas SCH94-03 or SCH94.32 hadsignificantly more CNS repair than control animals, measured by adetailed quantitative morphological assessment of CNS remyelination.Moreover, preliminary data suggest that the autoantibody, SCH94.03 isalso effective in preventing clinical relapses in mammals afflicted withexperimental autoimmune encephalomyelitis (EAE).

Clinical Disease in SJL/J Mice with Established R-EAE After Treatmentwith SCH94.03.

R-EAE was induced in SJL/J mice through adoptive transfer of MBP peptide(91-103)-specific T cells and treatment was initiated with monoclonalautoantibody SCH94.03, control IgM, or PBS after recovery from theinitial episode of clinical disease. Both the initial clinical diseasepeak and severity were similar between treatment groups (Table 4).However, treatment with SCH94.03 reduced the percentage of mice with afirst clinical relapse by half compared to mice treated with control IgMor PBS, and prolonged relapse onset by 6 days in those mice that didhave a clinical relapse. When only mice with severe initial clinicaldisease (score ≧3) were analyzed, 10 of 12 mice (83%) treated withcontrol IgM or PBS had a first relapse compared to only 3 of 9 mice(33%) treated with SCH94.03 (P<0.04 using a Fisher exact test),indicating that SCH94.03 was effective regardless of initial diseaseseverity. In addition, 4 mice treated with control IgM or PBSD had asecond clinical relapse, whereas no mouse treated with SCH94.03 had morethan one relapse, although this difference was not statisticallysignificant because of the few mice with a second relapse prior tosacrifice.

Spinal Cord Pathology in SJL/J Mice with Established R-EAE afterTreatment with SCH94.03.

Treatment with SCH94.03 also improved pathological disease inestablished R-EAE. Consistent with the reduction in clinical disease,treatment with SCH94.03d reduced by 40% both demyelination and meningealinflammation in the spinal cords of SJL/J mice with R-EAE (Table 5).Demyelinated lesions in mice treated with SCH94.03 were typicallysmaller in size with fewer inflammatory cells than mice treated withcontrol IgM or PBS. The majority of demyelinated lesions were located inthe dorsal columns in mice treated with SCH94.03 (57.0±5.4%; mean±SEM)and control IgM or PBS (51.5±4.8%; P>0.4 using a Student's t test). Theremainder of the demyelinated lesions in mice treated with SH94.03 orcontrol IgM or PBS were distributed between posterolateral (12.0±2.9%and 11.0±2.0%, respectively), anterolateral (14.3±3.0% and 20.3±2.5%),and ventral (14.9±17.1±1.6%) columns (P>0.1 for all).

To evaluate the relationship between clinical and pathological diseasein R-EAE, we correlated pathology scores (Table 5) with the severity ofthe initial clinical attack and any subsequent relapse (Table 4) inindividual mice. Regression analyses indicated a moderate butstatistically significant correlation between relapse severity and bothDemyelination®=0.64; P>0.6). These results suggest that in addition topreventing demyelination and meningeal inflammation, the overallclinical benefits of SCH94.03 were secondary to inhibition of diseaseprocesses not readily identifiable by standard pathological analysis.

TABLE 4 Clinical Disease in SJL/L Mice With R-EAE After Treatment WithSCH94.03 TREATMENT SCH94.03 Control* Number of Mice 14 19 Initial attackPeak (day)  13 ± 1‡  14 ± 1 Maximal clinical severity 2.8 ± 0.2‡ 2.8 ±0.2 First relapse No. mice relapsed (%) 5/14 (35.7)§ 15/19 (78.9) Onset¶2.4 ± 2**  18 ± 2 Maximal clinical severity 2.4 ± 2‡ 2.1 ± 0.2 SecondRelapse No. mice relapsed (%) 0/14 (0.0)‡‡  4/19 (21.1) Onset¶ —  29 ± 2Maximal clinical severity — 2.3 ± 0.4 Cumulative relapses  4 19 Lengthof follow-up (days)  56 ± 1‡  58 ± 1 SJL/J mice with R-EAE were injectedwith 50 μg SCH94.03, IgM, or an equivalent volume of PBS twice weeklyafter spontaneous recovery from the initial episode of clinical disease.Subsequent relapses were assessed and graded for severity the data are acomposite of 4 independent experiments and are presented as the means ±SEM where appropriate. *Combined data from mice treated with control IgM(n = 10) or PBS (n = 9). No differences were observed with any diseaseparameter between the two control groups. ‡Not significant (P > 0.05)when compared to control data using a Mann-Whitney rank sum test. §P <0.03 when compared to control data using a Fisher exact test. ¶Number ofdays from the peak of the initial attack. **P < 0.05 when compared tocontrol data using a Mann-Whitney rank sum test. ‡‡P = 0.12 whencompared to control data using a Fisher exact test.

TABLE 5 Pathological Disease in SJL/J Mice with R-EAE After Treatmentwith SCH 94.03 Pathological Score Meningeal Treatment n Demyelinationinflammation SCH94.03 14 24.6 ± 3.6* 18.7 ± 3.6* Control‡ 19 39.3 ± 6.0 31.8 ± 5.3  SJL/J mice with R-EAE were treated as described in the Table1 legend. The pathological scores were determined by a semi-quantitativemorphological analysis and represent the percentage of spinal cordquadrants with the indicated pathological abnormality. One mouse treatedwith control IgM had minimal gray matter inflammation, whereas all otheranimals shoed no inflammation in spinal cord gray matter. The data arefrom 4 independent experimented and are presented as the mean ± SEMwherend indicates the number of mice. *P < 0.05 when compared to controldata. ‡Combined data from mice treated with control IgM or PBS asdescribed in the Table 4 legend.

Thus, it is reasonable to predict that autoantibodies, such as SCH94.03,play a critical role in stopping an immune-mediated process ofdemyelination in CNS diseases.

Two potential mechanisms can be proposed by which Abs promoteremyelination. First, Abs might inhibit some pathogenic component of thedisease process, such as virus activity, an immune response whichdirectly suppresses remyelination. If the disease outcome is based upona balance between tissue destruction and repair, inhibition ofpathogenic components would allow a physiological repair response topredominate. Experimental and clinical evidence support this hypothesis.Spontaneous CNS remyelination is seen in MS patients and severalexperimental models of CNS demyelination as well as described herein,demonstrating spontaneous remyelination in control mice. This indicatesthat remyelination is a normal physiological response to myelin damage.In addition, treatment of mice chronically infected with TMEV withvarious immunosuppressive regiments promotes remyelination, but does notdecrease demyelination, indicating that there is an immunologicalcomponent which inhibits remyelination. Immunological function studiesreported in Miller et al., International Immunology, (1996) 8, pp131-141, the disclosure of which is incorporated herein by reference,indicate that animals treated with SCH94.03 had similar numbers of B andT (both CD4+ and CD8+) cells in their spleens compared to controlanimals, had similar in vitro splenocyte proliferative responses tomitogens and antigens, and mounted comparable Ab responses to both Tcell-dependent and T cell-independent antigens. See Table 6, below.However, there was a 2 to 3 fold reduction in the number of CD4 and CD8T cells infiltrating the CNS of mice treated with the mAb 94.03.Treatment with 94.03 also suppresses the humoral immune response to a Tcell-dependent antigen in chronically infected mice. Immuhistochemicalstaining showed that 94.03 labeled MHC Class II positive dendrite cellsin peripheral lymphoid organs. These results thus suggest that one ofthe mechanisms by which Mab SCH94.03 may be promoting remyelination isby inhibiting a pathogenic immune response.

TABLE 6 FCM analysis of mononuclear cells infiltrating the CNS ofchronically infected SJL/J mice. Total No. of surface marker positiveCNS- infiltrating mononuclear cells (×10⁻⁵)^(a) Treatment N CD5⁺ CD4⁺CD8⁺ CD45R(B220)⁺ PBS 10 6.2 ± 0.8 3.0 ± 0.4 2.4 ± 0.3 0.4 ± 0.1 ControlIgM 12 5.0 ± 0.6 3.0 ± 0.4 1.7 ± 0.2 0.2 ± 0.0 SCH94.03 12 2.3 ± 0.4^(b)1.4 ± 0.2^(c) 0.8 ± 0.2^(b) 0.1 ± 0.0^(d) SJL/J mice chronicallyinfected with TMEV were injected i.p. with a total dose of 0.5 mgSCH94.03, control IfgM or an equivalent volume PBS, divided into twiceweekly doses for 5 weeks. For control IgM, MOPC104E and XXMEN-OE5 wereused. The data are a composite of independent experiments and arepresented as the mean ± SEM, where N indicates the number of mice.^(a)Cell numbers were calculated by multiplying the percentage ofpositive cells assessed by FCM with the total number of mononuclearcells isolated from brain and spinal homogenates of individual mice byPercoll gradient separation. ^(b)P < 0.00001 when compared with combinedcontrol IgM and PBS data. ^(c)P < 0.00005 when compared with combinedcontrol IgM and PBS data. ^(d)P < 0.007 when compared with combinedcontrol IgM and PBS data.

The second hypothesis is that certain Abs can actively stimulate CNSremyelination, perhaps via stimulation of oligodendrocyte proliferationand/or differentiation in vivo, as has been demonstrated in vitro (Diaz,M. et al., Brain Res., 154:231-239 (1978); Raine, C. S., et al., Lab.Invest., 38:397-403 (1979); Lehrer, G. M. et al., Brain Res.,172:557-560 (1979); Bansal, R. et al., J. Neurosci. Res., 21:260-267(1988); Benjamins, J. A. and Dyer, C. A., Ann. NY Acad. Sci., 605:90-100(1990); Dyer, C. A., Mol. Neurobiol., 7:1-22 (1993)). MAb SCH94.03 maydirectly stimulate precursor glial cells which are known to be presentat the edges of both human and experimental CNS lesions which showactive remyelination. Alternatively, SCH94.03 may work indirectly, viaactivation of astrocytes or other accessory cells, which could releasefactors important for the survival or proliferation of cells in theoligodendroglial lineage. The formation of Ab-antigen complexes in situwith tissue components released upon myelin destruction may alsoparticipate in Ab-mediated CNS remyelination. Although SCH94.03 is notCNS-specific, the recognition of both surface and cytoplasmic antigenson glial cells by the mAb supports an active mechanism hypothesis. Incontrast to the immunomodulatory hypothesis, which would not necessarilyrequire that Abs has direct access to the CNS, the hypothesis that Absactively stimulate CNS remyelination implies the prerequisite of directaccess to the CNS. This is contrary to the view of the selectivepermeability of the blood-brain barrier, especially toward largemolecules such as pentameric IgM. However, during chronic inflammatoryconditions such as TMEV infection or MS, peripheral leukocytes migrateinto the CNS, indicating an alteration in the blood-brain barrierpermeability. Therefore, large proteins such as serum Ig might alsoenter, via either passive diffusion through “open” endothelium, orperhaps via an unidentified active transport mechanism.

Treatment of Demyelinating Diseases

The results of the experiments described herein have practicalapplications to multiple sclerosis (MS), EAE, and other related centralnervous system demyelinating disorders. Rare examples of spontaneousCNS-type remyelination (“shadow plaques”) are found in MS and occasionalperipheral nervous system (PNS)-type remyelination is found indemyelinated spinal cord plaques near the root entry zone.Oligodendrocytes are infrequent at the center of the chronic plaques inMS but they appear to proliferate at the periphery of plaques, wherethey are associated with abortive remyelination. The process ofremyelination may correlate with the spontaneous remission andimprovements observed clinically in MS. These clinical observationsindicate that new myelin formation is possible in MS. The remyelinationthat has been stimulated in mice with TMEV-induced demyelination byusing a mAb holds promise for therapeutic applications in multiplesclerosis.

Of importance clinically is the question of whether morphologicregeneration of thin myelin sheaths contributes to functional recovery.Computer simulations indicate that new myelin formation even byinappropriately thin sheaths improves impulse conduction. Since the axonmembrane of normally myelinated fibers is highly differentiated, it isnecessary for sodium channels to be present at high density at the nodeof Ranvier to propagate salutatory conduction. Experimental evidencesuggests that newly formed nodes do develop the required high sodiumchannel density as demonstrated by saxitoxin binding. Data to datesuggest that remyelination even by inappropriately thin myelin improvesconduction in a previously demyelinated axon. Therefore, any strategy topromote this morphologic phenomenon has the potential of producingfunctional recovery.

The data presented herein demonstrates, for the first time, thatadministration of a monoclonal antibody to a mammal is capable ofstimulating remyelination of central nervous system axons in vivo.Specifically, treatment of chronically infected TMEV-infected mice withas little as 10 μg of SCH94.03 resulted in a 4- to 5-fold increase inthe total area of CNS myelination compared to mice treated with acontrol mAb.

In addition, the isolation and testing of human autoantibodies,specifically polyclonal IgM antibodies and monoclonal antibodies, as setforth herein and particularly with reference to Examples 5-25 infra.supplements and enhances the advantages and capabilities of the presentinvention. Importantly, the use of human antibodies avoids the potentialfor human immune response against the therapeutic antibody. Therapeuticantibodies derived from non-human animals have been shown to generate animmune response, which can be significant and detrimental to theindividual. Accordingly, polyclonal human IgM and polyclonal human IgGhave been tested in two models of in vivo spinal cord demyelination; achronic viral infection model, and an acute toxicity model. In bothmodels polyclonal human IgM treated animals had a significantly higherdensity of newly myelinated axons than animals treated with polyclonalhuman IgG. A panel of human monoclonal IgM antibodies have also beenidentified, based on their reactivity with surface antigens specific tothe central nervous system. These human antibodies promote significantlymore central nervous system remyelination than polyclonal human IgG whengiven to mammals with demyelinating disease. The human monoclonalantibodies are antigenically polyreactive and recognize determinants onthe surface of oligodendrocytes and specific populations of neurons. Thelight and heavy chain variable regions of several human antibodies thatpromote remyelination have been sequenced. In particular, theseantibodies can induce calcium fluxes in glial cells (oligodendrocytesand astrocytes) in culture, suggestive of direct binding and signalingthrough glial cells. These human antibodies bind to human white matterand may be effective in promoting remyelination in humans. The benefitsof a monoclonal antibody for use as a therapeutic agent are 1) theantibody can be grown free of possible host infection and, 2) theantibody can be genetically altered in vitro to change itseffectiveness.

Thus, as a result of the experiments described herein, the method of thepresent invention can be used to treat mammals, including humans anddomestic animals, afflicted with demyelinating disorders, and tostimulate remyelination and regeneration of the CNS axons, as well as tooffer neuroprotection. As described herein, an effective amount of themonoclonal antibody or a peptide fragment, hapten, or equivalent, can beadministered by conventional routes of administration, and particularlyby, intravenous (iv) or intraperitoneal (ip) injection. As describedherein, therapeutic compositions and vaccines are contemplated and maybe prepared and administered. An effective amount of the antibody canvary depending on the size of the mammal being treated, the severity ofthe disease, the route of administration, and the course of treatment.For example, each dose of antibody administered can range fromapproximately 0.5 mg/kg to approximately 400 mg/kg, with the preferredrange from approximately 0.5 mg/kg to approximately 250 mg/kg. It isimportant to note that a dose of mAb as low as 10 μg (0.5 mg/kg) waseffective in promoting remyelination of CNS axons in mice. The dose ofantibody will also depend on the route of administration. For example,an iv dose administered to mice was 0.5 mg/kg, and an ip dose was 5.0mg/kg. The course of treatment includes the frequency of administrationof the antibody (e.g., daily, weekly, or bi-weekly) and the duration ofthe treatment (e.g., four weeks to four months). Thus, for example, alarger amount of mAb can be given daily for four to five weeks, asopposed to a smaller amount of mAb given for four months.

The effectiveness of the amount of the monoclonal antibody beingadministered can be assessed using any number of clinical criteria, forexample, as described in the Examples herein, including overallappearance of the mammal, the activity of the mammal and the extent ofparalysis of the mammal. The effectiveness of the amount of monoclonalantibody necessary to induce remyelination in humans can also beassessed in a double blinded controlled trial. Patients with fixedneurological deficits from demyelinating disease can be treated withmonoclonal antibody or controls. Improvement in isometric musclestrength as detected by quantitative biomechanics muscle testing couldbe used as the primary therapeutic end-point.

Additionally, the monoclonal antibody may be genetically altered, e.g.“humanized” by the substitution of human antibody nucleotide sequencesin non-variable regions of the murine mAb to reduce immunogenicity.

In addition to in vivo methods of promoting remyelination, ex vivomethods of stimulating remyelination in CNS axons are also encompassedby the present invention. For example, the monoclonal antibody may beused in vitro to stimulate the proliferation and/or differentiation ofglial cells, such as oligodendrocytes, as described e.g. in Example 2and Example 17. These exogenous glial cells can then be introduced intothe CNS of mammals using known techniques. Remyelination of CNS axonswould be increased by increasing the number of endogenous glial cellspresent (glial cells, such as oligodendrocytes play a critical role inthe production of myelin).

In vitro methods of producing glial cells, or stimulating theproliferation of glial cells from mixed culture (e.g., rat optic nervecell, or rat brain cell cultures) are also encompassed by thisinvention. For example, cells obtained from rat optic nerve, or ratbrain, containing glial cells, are cultured as a mixed culture underconditions sufficient to promote growth of the cells. An effectiveamount of mAb capable of promoting remyelination of CNS axons, such asSCH94.03, sHIgM22 or sHIgM46, or a combination thereof, is then added tothe mixed culture of cells and maintained under conditions sufficientfor growth and proliferation of cells. The mAb stimulates theproliferation of glial cells cultured in the presence of the mAb isincreased, relative to the proliferation of glial cells grown in theabsence of the mAb.

As stated above, the antibodies for use in the methods of the presentinvention can be, and are preferably, administered as medicaments, i.e.,pharmaceutical compositions. An effective amount of the polyclonal IgMantibody can thus be combined with, or diluted with, an appropriatepharmaceutically acceptable carrier, diluent or vehicle, such as aphysiological buffer or saline solution. An effective amount of themonoclonal antibody can thus be combined with, or diluted with, anappropriate pharmaceutically acceptable carrier, diluent or vehicle,such as a physiological buffer, or saline solution. An effective amountof a combination of one or more monoclonal antibody may be similarlycombined with or diluted with an appropriate pharmaceutically acceptablecarrier, diluent or vehicle. In the instance where a vaccine is to beprepared, the monoclonal antibody or equivalent active of the inventionmay be prepared with a pharmaceutically effective and suitable carrieror adjuvant, and the protocol for administration may proceed inaccordance with standard procedures for immunization known to theskilled practitioner.

The pharmaceutical compositions used in the methods of this inventionfor administration to animals and humans comprise the polyclonal IgMantibodies or monoclonal antibodies in combination with a pharmaceuticalcarrier or excipient. In a preferred embodiment, the pharmaceuticalcomposition may contain more than one, preferably two, monoclonalautoantibodies of the present invention. Thus, pharmaceuticalcompositions comprising, for example, an effective amount in combinationof sHIgM22 and sHIgM46 are contemplated herein. Such compositions areadvantageous in that the presence of more than one monoclonalautoantibody will potentiate the activity of others in the sametherapeutic composition or method.

The medicament can be in the form of tablets (including lozenges andgranules), dragees, capsules, pills, ampoules or suppositoriescomprising the compound of the invention.

Advantageously, the compositions are formulated as dosage units, eachunit being adapted to supply a fixed dose of active ingredients.Tablets, coated tablets, capsules, ampoules and suppositories areexamples of preferred dosage forms according to the invention. It isonly necessary that the active ingredient constitute an effectiveamount, i.e., such that a suitable effective dosage will be consistentwith the dosage form employed in single or multiple unit doses. Theexact individual dosages, as well as daily dosages, will, of course, bedetermined according to standard medical principles under the directionof a physician or veterinarian.

The monoclonal antibodies can also be administered as suspensions,solutions and emulsions of the active compound in aqueous or non-aqueousdiluents, syrups, granulates or powders.

Diluents that can be used in pharmaceutical compositions (e.g.,granulates) containing the active compound adapted to be formed intotablets, dragees, capsules and pills include the following: (a) fillersand extenders, e.g., starch, sugars, mannitol and silicic acid; (b)binding agents, e.g., carboxymethyl cellulose and other cellulosederivatives, alginates, gelatine and polyvinyl pyrrolidone; (c)moisturizing agents, e.g., glycerol; (d) disintegrating agents, e.g.,agar-agar, calcium carbonate and sodium bicarbonate; (e) agents forretarding dissolution, e.g., paraffin; (f) resorption accelerators,e.g., quaternary ammonium compounds; (g) surface active agents, e.g.,cetyl alcohol, glycerol monostearate; (g) adsorptive carriers, e.g.,kaolin and bentonite; (i) lubricants, e.g., talc, calcium and magnesiumstearate and solid polyethylene glycols.

The tablets, dragees, capsules and pills comprising the active compoundcan have the customary coatings, envelopes and protective matrices,which may contain opacifiers. They can be so constituted that theyrelease the active ingredient only or preferably in a particular part ofthe intestinal tract, possibly over a period of time. The coatings,envelopes and protective matrices may be made, for example, frompolymeric substances or waxes.

The diluents to be used in pharmaceutical compositions adapted to beformed into suppositories can, for example, be the usual water-solublediluents, such as polyethylene glycols and fats (e.g., cocoa oil andhigh esters, [e.g., C₁₄-alcohol with C₁₆-fatty acid]) or mixtures ofthese diluents.

The pharmaceutical compositions which are solutions and emulsions can,for example, contain the customary diluents (with, of course, theabove-mentioned exclusion of solvents having a molecular weight below200, except in the presence of a surface-active agent), such assolvents, dissolving agents and emulsifiers. Specific non-limitingexamples of such diluents are water, ethyl alcohol, isopropyl alcohol,ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (forexample, ground nut oil, glycerol, tetrahydrofurfuryl alcohol,polyethylene glycols and fatty acid esters of sorbitol or mixturesthereof.

For parental administration, solutions and suspensions should besterile, e.g., water or arachis oil contained in ampoules and, ifappropriate, blood-isotonic.

The pharmaceutical compositions which are suspensions can contain theusual diluents, such as liquid diluents, e.g., water, ethyl alcohol,propylene glycol, surface active agents (e.g., ethoxylated isostearylalcohols, polyoxyethylene sorbitols and sorbitan esters),microcrystalline cellulose, aluminum methahydroxide, bentonite,agar-agar and tragacanth, or mixtures thereof.

The pharmaceutical compositions can also contain coloring agents andpreservatives, as well as perfumes and flavoring additions (e.g.,peppermint oil and eucalyptus oil), and sweetening agents, (e.g.,saccharin and aspartame).

The pharmaceutical compositions will generally contain from 0.5 to 90%of the active ingredient by weight of the total composition.

In addition to the monoclonal antibodies, the pharmaceuticalcompositions and medicaments can also contain other pharmaceuticallyactive compounds, e.g. steroids, anti-inflammatory agents or the like.

Any diluent in the medicaments of the present invention may be any ofthose mentioned above in relation to the pharmaceutical compositions.Such medicaments may include solvents of molecular weight less than 200as the sole diluent.

It is envisaged that the polyclonal IgM antibodies and monoclonalantibodies will be administered perorally, parenterally (for example,intramuscularly, intraperitoneally, subcutaneously, transdermally orintravenously), rectally or locally, preferably orally or parenterally,especially perlingually, or intravenously.

The administered dosage rate will be a function of the nature and bodyweight of the human or animal subject to be treated, the individualreaction of this subject to the treatment, type of formulation in whichthe active ingredient is administered, the mode in which theadministration is carried out and the point in the progress of thedisease or interval at which it is to be administered. Thus, it may insome case suffice to use less than a minimum dosage rate, while othercases an upper limit must be exceeded to achieve the desired results.Where larger amounts are administered, it may be advisable to dividethese into several, individual administrations over the course of theday.

According to the invention, the component or components of a therapeuticcomposition of the invention may be introduced parenterally,intrathecally, transmucosally, e.g., orally, nasally, pulmonarally, orrectally, or transdermally. Preferably, administration is parenteral,e.g., via intravenous injection, and also including, but is not limitedto, intra-arterial, intramuscular, intradermal, subcutaneous,intraperitoneal, intraventricular, and intracranial administration. Oralor pulmonary delivery may be preferred to activate mucosal immunity;since the bacteria responsible for the conditions under treatmentgenerally colonize the nasopharyngeal and pulmonary mucosa, mucosaladministration may be particularly effective as a treatment. The term“unit dose” when used in reference to a therapeutic composition of thepresent invention refers to physically discrete units suitable asunitary dosage for humans, each unit containing a predetermined quantityof active material calculated to produce the desired therapeutic effectin association with the required diluent; i.e., carrier, or vehicle.

In another embodiment, the active compound can be delivered in avesicle, in particular a liposome (see Langer, Science 249:1527-1533(1990); Treat et al., in Liposomes in the Therapy of Infectious Diseaseand Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp.353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generallyibid).

M yet another embodiment, the therapeutic compound can be delivered in acontrolled release system. For example, the polypeptide may beadministered using intravenous infusion, an implantable osmotic pump, atransdermal patch, liposomes, or other modes of administration. In oneembodiment, a pump may be used (see Langer, supra; Sefton, CRC Crit.Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980);Saudek et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment,polymeric materials can be used (see Medical Applications of ControlledRelease, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974);Controlled Drug Bioavailability, Drug Product Design and Performance,Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J.Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al.,Science 228:190 (1985); During et al., Ann. Neurol. 25:351 (1989);Howard et al., J. Neurosurg. 71:105 (1989)). In yet another embodiment,a controlled release system can be placed in proximity of thetherapeutic target, i.e., the brain, thus requiring only a fraction ofthe systemic dose (see, e.g., Goodson, in Medical Applications ofControlled Release, supra, vol. 2, pp. 115-138 (1984)). Preferably, acontrolled release device is introduced into a subject in proximity ofthe site of inappropriate immune activation or a tumor. Other controlledrelease systems are discussed in the review by Langer (Science249:1527-1533 (1990)).

A subject in whom administration of an active component as set forthabove is an effective therapeutic regimen for a condition or pathologyassociated with the central nervous system, including in certaininstances, bacterial infection is preferably a human, but can be anyanimal. Thus, as can be readily appreciated by one of ordinary skill inthe art, the methods and pharmaceutical compositions of the presentinvention are particularly suited to administration to any animal,particularly a mammal, and including, but by no means limited to,domestic animals, such as feline or canine subjects, farm animals, suchas but not limited to bovine, equine, caprine, ovine, and porcinesubjects, wild animals (whether in the wild or in a zoological garden),research animals, such as mice, rats, rabbits, goats, sheep, pigs, dogs,cats, etc., i.e., for veterinary medical use.

In the therapeutic methods and compositions of the invention, atherapeutically effective dosage of the active component is provided. Atherapeutically effective dosage can be determined by the ordinaryskilled medical worker based on patient characteristics (age, weight,sex, condition, complications, other diseases, etc.), as is well knownin the art. Furthermore, as further routine studies are conducted, morespecific information will emerge regarding appropriate dosage levels fortreatment of various conditions in various patients, and the ordinaryskilled worker, considering the therapeutic context, age and generalhealth of the recipient, is able to ascertain proper dosing. Generally,for intravenous injection or infusion, dosage may be lower than forintraperitoneal, intramuscular, or other route of administration. Thedosing schedule may vary, depending on the circulation half-life, andthe formulation used. The compositions are administered in a mannercompatible with the dosage formulation in the therapeutically effectiveamount. Precise amounts of active ingredient required to be administereddepend on the judgment of the practitioner and are peculiar to eachindividual. However, suitable dosages may range from about 0.1 to 20,preferably about 0.5 to about 10, and more preferably one to several,milligrams of active ingredient per kilogram body weight of individualper day and depend on the route of administration. Suitable regimes forinitial administration and booster shots are also variable, but aretypified by an initial administration followed by repeated doses at oneor more hour intervals by a subsequent injection or otheradministration. Alternatively, continuous intravenous infusionsufficient to maintain concentrations of ten nanomolar to ten micromolarin the blood are contemplated.

Administration with other compounds. For treatment of a demyelinatingcondition, for instance multiple sclerosis, one may administer thepresent active component in conjunction with one or more pharmaceuticalcompositions used for treating multiple sclerosis, including but notlimited to (1) anti-inflammatory agents, such as steroids; (2)Betaseron; (3) Copaxone; or 94) polyclonal IgM. Administration may besimultaneous (for example, administration of a mixture of the presentactive component and an antibiotic), or may be in seriatim.

Accordingly, in specific embodiment, the therapeutic compositions mayfurther include an effective amount of the active component, and one ormore of the following active ingredients: an antibiotic, a steroid, etc.

Also contemplated herein is pulmonary delivery of the presentneuromodulatory agent or agents, which may be associated with ananti-inflammatory. Reports of preparation of proteins for pulmonarydelivery are found in the art [Adjei et al. Pharmaceutical Research,7:565-569 (1990); Adjei et al., International Journal of Pharmaceutics,63:135-144 (1990) (leuprolide acetate); Braquet et al., Journal ofCardiovascular Pharmacology, 13(suppl. 5):143-146 (1989) (endothelin-1);Hubbard et al., Annals of Internal Medicine, Vol. III, pp. 206-212(1989) (al-antitrypsin); Smith et al., J. Clin. Invest. 84:1145-1146(1989) (α-1-proteinase); Oswein et al., “Aerosolization of Proteins”,Proceedings of Symposium on Respiratory Drug Delivery II, Keystone,Colo., March, (1990) (recombinant human growth hormone); Debs et al., J.Immunol. 140:3482-3488 (1988) (interferon-γ and tumor necrosis factoralpha); Platz et al., U.S. Pat. No. 5,284,656 (granulocyte colonystimulating factor)]. A method and composition for pulmonary delivery ofdrugs is described in U.S. Pat. No. 5,451,569, issued Sep. 19, 1995 toWong et al.

All such devices require the use of formulations suitable for thedispensing of adhesin inhibitory agent (or derivative). Typically, eachformulation is specific to the type of device employed and may involvethe use of an appropriate propellant material, in addition to the usualdiluents, adjuvant and/or carriers useful in therapy. Also, the use ofliposomes, microcapsules or microspheres, inclusion complexes, or othertypes of carriers is contemplated. Chemically modified adhesininhibitory agent may also be prepared in different formulationsdepending on the type of chemical modification or the type of deviceemployed.

Formulations suitable for use with a nebulizer, either jet orultrasonic, will typically comprise neuromodulatory agent (orderivative) dissolved in water at a concentration of about 0.1 to 25 mgof biologically active agent per ml of solution. The formulation mayalso include a buffer and a simple sugar (e.g., for neuromodulatoryagent stabilization and regulation of osmotic pressure). The nebulizerformulation may also contain a surfactant, to reduce or prevent surfaceinduced aggregation of the neuromodulatory agent caused by atomizationof the solution in forming the aerosol.

Formulations for use with a metered-dose inhaler device will generallycomprise a finely divided powder containing the neuromodulatory agent(or derivative) suspended in a propellant with the aid of a surfactant.The propellant may be any conventional material employed for thispurpose, such as a chlorofluorocarbon, a hydrochlorofluorocarbon, ahydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane,dichlorodifluoromethane, dichlorotetrafluoroethanol, and1,1,1,2-tetrafluoroethane, or combinations thereof. Suitable surfactantsinclude sorbitan trioleate and soya lecithin. Oleic acid may also beuseful as a surfactant.

The liquid aerosol formulations contain neuromodulatory agent and adispersing agent in a physiologically acceptable diluent. The dry powderaerosol formulations of the present invention consist of a finelydivided solid form of neuromodulatory agent and a dispersing agent. Witheither the liquid or dry powder aerosol formulation, the formulationmust be aerosolized. That is, it must be broken down into liquid orsolid particles in order to ensure that the aerosolized dose actuallyreaches the mucous membranes of the nasal passages or the lung. The term“aerosol particle” is used herein to describe the liquid or solidparticle suitable for nasal or pulmonary administration, i.e., that willreach the mucous membranes. Other considerations, such as constructionof the delivery device, additional components in the formulation, andparticle characteristics are important. These aspects of pulmonaryadministration of a drug are well known in the art, and manipulation offormulations, aerosolization means and construction of a delivery devicerequire at most routine experimentation by one of ordinary skill in theart. In a particular embodiment, the mass median dynamic diameter willbe 5 micrometers or less in order to ensure that the drug particlesreach the lung alveoli [Wearley, L. L., Crit. Rev. in Ther. Drug CarrierSystems 8:333 (1991)].

The neuromodulatory agents of the invention may also be prepared foradministration in the form of vaccines, which may comprise as theactive, the herein recited autoantibodies, peptide analogs, or haptens,or possibly combinations thereof. Thus, the preparation of vaccines mayproceed in accordance with known procedures, and monovalent as well aspolyvalent vaccines are contemplated. Also, DNA sub unit vaccines, basedupon the DNA molecules of the invention, may be prepared. All vaccinesmay be administered in accordance with standard practices of thephysician or clinician, and such parameters are considered to be withinthe scope of the present invention.

Vectors containing e.g. a DNA-based vaccine in accordance with theinvention can be introduced into the desired host by methods known inthe art, e.g., transfection, electroporation, microinjection,transduction, cell fusion, DEAE dextran, calcium phosphateprecipitation, lipofection (lysosome fusion), use of a gene gun, or aDNA vector transporter (see, e.g., Wu et al., 1992, J. Biol. Chem.267:963-967; Wu and Wu, 1988, J. Biol. Chem. 263:14621-14624; Hartmut etal., Canadian Patent Application No. 2,012,311, filed Mar. 15, 1990).

The vaccine can be administered via any parenteral route, including butnot limited to intramuscular, intraperitoneal, intravenous, and thelike. Preferably, since the desired result of vaccination is toelucidate an immune response to the antigen, and thereby to thepathogenic organism, administration directly, or by targeting or choiceof a viral vector, indirectly, to lymphoid tissues, e.g., lymph nodes orspleen, is desirable. Since immune cells are continually replicating,they are ideal target for retroviral vector-based nucleic acid vaccines,since retroviruses require replicating cells.

Passive immunity can be conferred to an animal subject suspected ofsuffering an autoimmune-mediated demyelinating disease, e.g. multiplesclerosis, by administering antiserum, polyclonal antibodies, or aneutralizing monoclonal antibody to the patient. Preferably, theantibodies administered for passive immune therapy are autologousantibodies. For example, if the subject is a human, preferably theantibodies are of human origin or have been “humanized,” in order tominimize the possibility of an immune response against the antibodies.The active or passive vaccines of the invention, or the administrationof an adhesin, can be used to protect an animal subject from infectionof a Gram positive bacteria, preferably streptococcus, and morepreferably, pneumococcus.

Further, the present invention contemplates treatment by gene therapy,where the appropriate neuromodulatory agent is correspondinglyintroduced to target cells for treatment, to cause or increaseexpression of the corresponding agent. Thus, in one embodiment, the DNAor a gene encoding the neuromodulatory agent, autoantibody, antibodypeptide, etc., or a protein or polypeptide domain fragment thereof isintroduced in vivo, ex vivo, or in vitro using a viral vector or throughdirect introduction of DNA. Expression in targeted tissues can beeffected by targeting the transgenic vector to specific cells, such aswith a viral vector or a receptor ligand, or by using a tissue-specificpromoter, or both.

Viral vectors commonly used for in vivo or ex vivo targeting and therapyprocedures are DNA-based vectors and retroviral vectors. Methods forconstructing and using viral vectors are known in the art [see, e.g.,Miller and Rosman, BioTechniques 7:980-990 (1992)].

DNA viral vectors include an attenuated or defective DNA virus, such asbut not limited to herpes simplex virus (HSV), papillomavirus, EpsteinBarr virus (EBV), adenovirus, adeno-associated virus (AAV), and thelike. Defective viruses, which entirely or almost entirely lack viralgenes, are preferred. Defective virus is not infective afterintroduction into a cell. Use of defective viral vectors allows foradministration to cells in a specific, localized area, without concernthat the vector can infect other cells. Thus, adipose tissue can bespecifically targeted. Examples of particular vectors include, but arenot limited to, a defective herpes virus 1 (HSV1) vector [Kaplitt etal., Molec. Cell. Neurosci. 2:320-330 (1991)], defective herpes virusvector lacking a glyco-protein L gene [Patent Publication RD 371005 A],or other defective herpes virus vectors [International PatentPublication No. WO 94/21807, published Sep. 29, 1994; InternationalPatent Publication No. WO 92/05263, published Apr. 2, 1994]; anattenuated adenovirus vector, such as the vector described byStratford-Perricaudet et al. [J. Clin. Invest. 90:626-630 (1992); seealso La Salle et al., Science 259:988-990 (1993)]; and a defectiveadeno-associated virus vector [Samulski et al., J. Virol. 61:3096-3101(1987); Samulski et al., J. Virol. 63:3822-3828 (1989); Lebkowski etal., Mol. Cell. Biol. 8:3988-3996 (1988)].

Preferably, for in vivo administration, an appropriate immunosuppressivetreatment is employed in conjunction with the viral vector, e.g.,adenovirus vector, to avoid immuno-deactivation of the viral vector andtransfected cells. For example, immunosuppressive cytokines, such asinterleukin-12 (IL-12), interferon-γ (IFN-γ), or anti-CD4 antibody, canbe administered to block humoral or cellular immune responses to theviral vectors [see, e.g., Wilson, Nature Medicine (1995)]. In addition,it is advantageous to employ a viral vector that is engineered toexpress a minimal number of antigens.

In another embodiment the DNA or gene can be introduced in a retroviralvector, e.g., as described in Anderson et al., U.S. Pat. No. 5,399,346;Mann et al., 1983, Cell 33:153; Temin et al., U.S. Pat. No. 4,650,764;Temin et al., U.S. Pat. No. 4,980,289; Markowitz et al., 1988, J. Virol.62:1120; Temin et al., U.S. Pat. No. 5,124,263; International PatentPublication No. WO 95/07358, published Mar. 16, 1995, by Dougherty etal.; and Kuo et al., 1993, Blood 82:845. Retroviral vectors can beconstructed to function as infections particles or to undergo a singleround of transfection. In the former case, the virus is modified toretain all of its genes except for those responsible for oncogenictransformation properties, and to express the heterologous gene.Non-infectious viral vectors are prepared to destroy the viral packagingsignal, but retain the structural genes required to package theco-introduced virus engineered to contain the heterologous gene and thepackaging signals. Thus, the viral particles that are produced are notcapable of producing additional virus.

Targeted gene delivery is described in International Patent PublicationWO 95/28494, published October 1995.

Alternatively, the vector can be introduced in vivo by lipofection. Forthe past decade, there has been increasing use of liposomes forencapsulation and transfection of nucleic acids in vitro. Syntheticcationic lipids designed to limit the difficulties and dangersencountered with liposome mediated transfection can be used to prepareliposomes for in vivo transfection of a gene encoding a marker [Feigner,et. al., Proc. Natl. Acad. Sci. U.S.A. 84:7413-7417 (1987); see Mackey,et al., Proc. Natl. Acad. Sci. U.S.A. 85:8027-8031 (1988); Ulmer et al.,Science 259:1745-1748 (1993)]. The use of cationic lipids may promoteencapsulation of negatively charged nucleic acids, and also promotefusion with negatively charged cell membranes [Feigner and Ringold,Science 337:387-388 (1989)]. The use of lipofection to introduceexogenous genes into the specific organs in vivo has certain practicaladvantages. Molecular targeting of liposomes to specific cellsrepresents one area of benefit. It is clear that directing transfectionto particular cell types would be particularly advantageous in a tissuewith cellular heterogeneity, such as pancreas, liver, kidney, and thebrain. Lipids may be chemically coupled to other molecules for thepurpose of targeting [see Mackey, et. al., supra]. Targeted peptides,e.g., hormones or neurotransmitters, and proteins such as antibodies, ornon-peptide molecules could be coupled to liposomes chemically.

It is also possible to introduce the vector in vivo as a naked DNAplasmid. Naked DNA vectors for gene therapy can be introduced into thedesired host cells by methods known in the art, e.g., transfection,electroporation, microinjection, transduction, cell fusion, DEAEdextran, calcium phosphate precipitation, use of a gene gun, or use of aDNA vector transporter [see, e.g., Wu et al., J. Biol. Chem. 267:963-967(1992); Wu and Wu, J. Biol. Chem. 263:14621-14624 (1988); Hartmut etal., Canadian Patent Application No. 2,012,311, filed Mar. 15, 1990;Williams et al., Proc. Natl. Acad. Sci. USA 88:2726-2730 (1991)].Receptor-mediated DNA delivery approaches can also be used [Curiel etal., Hum. Gene Ther. 3:147-154 (1992); Wu and Wu, J. Biol. Chem.262:4429-4432 (1987)].

In a preferred embodiment of the present invention, a gene therapyvector as described above employs a transcription control sequence thatcomprises the DNA consensus sequence recognized by e.g. an autoantibodyof the invention, i.e., an antibody binding site, operably associatedwith a therapeutic heterologous gene inserted in the vector. That is, aspecific expression vector of the invention can be used in gene therapy.

The present invention will be better understood from a consideration ofthe following non-limiting examples, which describe the preparation ofmaterials, compounds and compositions and the development and practiceof methods illustrative of the present invention. It will be apparent tothose skilled in the art that many modifications, both of materials andmethods, may be practiced without departing from the purpose and intentof this disclosure. The following examples are presented in order tomore fully illustrate the preferred embodiments of the invention andserve also in fulfillment of applicants' duty to present the best modeknown for the practice of the invention, and should in no way beconstrued as limiting the broad scope thereof.

EXAMPLES Example 1

Monoclonal Antibody Production, Screening and Purification

Animals

Spleens of two SJL/J mice (Jackson Laboratories, Bar Harbor, Me.) thathad been injected twice with spinal cord homogenate (SCH) in incompleteFreund's adjuvant were used as the source of B cells for fusion andhybridoma production. Splenocytes were fused with NS-1 myeloma cellsusing polyethylene glycol, and viable cell fusions were selected withhypoxanthine-aminopterin-thymidine (HAT) media and cloned by limitingdilution as described (Katzmann, J. A. et al., Proc. Nat. Acad. Sci.USA, 78:162-166 (1981)).

ELISAs

Hybridoma supernatants from viable Ig-producing clones were screened forbinding to SCH by an enzyme-linked immunosorbant assay (ELISA). Thefollowing antigens were used for screening mAbs: SCH—(10 μg)reconstituted in carbonate-bicarbonate buffer (pH 8.53), MBP—(1 μg)dissolved in PBS, GC (1 μg) dissolved in absolute alcohol, PLP (1 μg)dissolved in water. PLP was provided by Dr. W. Macklin (UCLS) who haspublished a solid phase immunoassay for PLP. For SCH, MBP or GC ELISA,Immuno II plates were coated with prepared antigen (100 μl/well) whichwas incubated overnight at 4° C. The following day well were washed inPBS and blocked with PBS+1% serum for 1 hour at room temperature. Plateswere washed again in PBS and serial dilutions of primary Ab diluted inPBS/0.1% BSA were added and incubated at room temperature for 2 hours.Plates were washed in PBS/0.05% Tween and appropriate secondary Abconjugated to alkaline phosphatase (1:1000 in PBS 0.1% BSA) was added.Plates were incubated at 37° C. for 2 hours, washed in PBS 0.05% Tween,and the substrate (Sigma 104 Phosphatase Substrate Tablet in 5 mldiethanolamine buffer) was added for 30 min. The reaction was terminatedwith 50 μl of 1 N NaOH. The plates were read on a Dynatech ELISA platereader.

Ascites Production

The hybridomas chosen for treatment experiments were injected intopristane-treated BALB/c mice for ascites production. Hybridomas werealso grown in RPM1-1640 media supplemented with 10% fetal bovine serumfor IgM production. IgM mAbs were purified by either ammonium sulfateprecipitation and gel filtration on a Sephacryl S-400 HR (Sigma) columnfor the initial transfer experiments, or by affinity chromatographyusing goat anti-mouse IgM (μ-chain specific; Jackson Immunoresearch,West Grove, Pa.) coupled to Reacti-Gel 6× matrix (Pierce, Rockford,Ill.) for later transfer experiments.

Example 2

In Vitro Testing of Monoclonal Antibodies

Selection of mABs that Promote Glial Cell Proliferation

The ability of the mAbs to promote proliferation of glial cells in vitrowas tested. Glial cells isolated from rat brain or optic nerves wereseeded in Falcon Microtest II plates at a concentration of 2×10₄ cellsper well in 0.1 ml of DME. Whole serum (SCH, IFA, MBP, GC, MBP/GC, PBSor PLP), purified Ig or mAb, was serially diluted and 0.1 ml aliquot wasadded to cells and assayed in triplicate. Three days later ³H-thymidinewas added (1 μCi/ml) and cells were harvested after 17 hours with anautomated cell harvester (Mash II Harvester). To document identity ofcells proliferating (i.e., astrocytes, progenitor glial cells,macrophages), selected cultures after exposure to ³H-thymidine, wereincubated with appropriate Ab specific for cell type followed by ABCimmunoperoxidase technique. After reaction of Hanker-Yates reagent, theslides were immersed in Ilford K2 nuclear emulsions, exposed for 4 daysat 4° C. and developed.

mAb 94.03 and 94.32 induce proliferation of mixed rat optic nerve braincultures

One- to two-day-old rats were killed with ether. Through carefuldissection, optic nerves were removed from the optic nerve chiasm to theeye. Nerves were transferred to centrifuge tubes containing 2 mis ofDMEM. An equal volume of 0.25% trypsin was added and incubated to 37° C.in a water bath for 45 min. 0.2 ml of FCS was added to terminatetrypsinization. Nerves were passed through a sterile needle and syringe(gauge no. 21) and then centrifuged at 1400 rpm for 10 minutes. The cellcount was adjusted to provide concentration of 5×10⁵ cells/100 μl ofmedia in 24-well trays in DMEM+0.5% FCS. After 12 to 16 hours,appropriate antibodies or growth media were added as per experimentalprotocols.

Brains of 1-2 day old rats were removed and placed in Hank's BalancedSalt Solution with 10 mM HEPES buffer (HBSS/H), approximately 1-2 ml perbrain. The brain stem, cerebellum, nd midbrain was discarded whereas theforebrain was minced with a bent syringe. The tissue was furtherdisrupted by repeated passage through a 10 ml pipet and transferred to a50 ml conical tube. The tissue suspension was shaken on a rotary shaker(75 rpm) for 30 min at 37° C. Trypsin was added to a final concentrationof 0.125% and the suspension was shaken for an additional 60 minutes.Trypsin digestion was stopped by adding FCS (10%). The cell suspensionwas passed sequentially through 120 and 54 μm Nytex, centrifuged,resuspended in serum-free medium with 10% FCS, and filtered againthrough 54 μm Nytex. Serum-free media was DMEM with 3.7 g/l sodiumbicarbonate, 6.0 g/l glucose, 2 mM L-glutamine, 0.1 nM nonessentialamino acids, 5 μg/ml insulin, 5 μg/ml streptomycin. The cells werecounted, plated onto uncoated tissue culture flasks or plates at 5×10₄cells/cm₂ and cultured at 37° C. in 5% CO₂. The media was changed after72 hours, and every 48 hours thereafter. On day 8 after cultureinitiation, the media was aspirated and replaced by SFM with varioussupplements (for example, antibody). For most experiments, the cellswere grown for an additional 48 hours before harvesting. Cells werepulsed with [³H]thymidine (5 μCi/ml) for the final 1824 hours ofculture.

Western Blot Procedure

Antigens were denatured and solubilized by heating at 100° C. in sodiumdodecyl sulfate (SDS) sample buffer. Samples were electrophoresed onstacking and separating gels containing 4.75% and 12.0% acrylamide at200 volts. After electrophoresis, gels and nitrocellulose membranes wereequilibrated for 30 minutes in transfer buffer (25 mM Tris, 192 mMglycine, 20% methanol, pH 8.1-8.3). All steps were done at roomtemperature. Gels were electroblotted for either 1 hour at 100V orovernight at 30V using the Bio-Rad Mini Trans-blot apparatus. Thenitrocellulose membrane was cut into strips and washed, 3×TBS (100 mMNaCl, 50 mM Trig, pH 7.6) with 0.03% Tween 20. Nitrocellulose stripswere blocked (TBS with 3% non-fat milk and 0.03% Tween 20) for 2-4hours, washed 3×, and incubated with primary Ab or antisera (diluted inblocking buffer) for 4 hours or overnight. After primary Ab incubation,strips were washed 3×, incubated with either biotin- or alkalinephosphate-labeled secondary Ab (diluted in blocking buffer) for 2 hours,washed 3×, and incubated with alkaline-phosphatase labeled-streptavidin(diluted in blocking buffer) for 2 hours if the biotin system is used.Nitrocellulose strips were washed 4× (final wash in TBS without Tween20) and incubated with substrate solution (0.165 mg/ml BCIP and 0.33mg/ml NBT in 100 mM sodium chloride, 100 mM Tris, 5 mM MgG12, pH 9.5)until sufficient color developed (approximately 10-15 min). The reactionwas stopped by adding PBS with 5 mM EDTA.

Cell lines or mixed brain cultures were lysed in 1×SDS reducing samplebuffer (2.3% SDS, 10% 2-ME, 0.125 M Tris, 20% glycerol) and heated to85° C. for 15 minutes. Nucleic acids were sheared by repeated passage oflysate through 21-27-gauge needles. Lysate proteins were separated on a12% acrylamide reducing gel, transferred to nitrocellulose membranes,and blotted with various antibodies as previously described.

Example 3

Promotion of CNS Remyelination Using a Monoclonal Antibody

Virus

The DA strain of TMEV was obtained from Drs. J. Lehrich and B. Amasonafter eight passages in BHK cells. The virus was passaged an additionalfour times at a multiplicity of infection of 0.1 plaque forming units(PFU) per cell. Cell-associated virus was released by Freeze-thawing thecultures followed by sonication. The lysate was clarified bycentrifugation and stored in aliquots at −70° C. All subsequentexperiments will use passage 12 virus. This virus isolate causes whitematter pathology without destruction of anterior horn cells.

In Vitro TMEV Neutralization Assay

Viral plaque assays were done as previously described (Patick, A. K., etal., J. Neuropath. Exp. Neurol., 50:523-537 (1991)). To assessneutralization, aliquots of TMEV (200 PFU/ml) were incubated withvarious concentrations of Ab for 1 hour t room temperature prior toplating onto confluent L2 cells. As a positive control, serum fromsusceptible mice chronically infected with TMEV was used. Under theassay conditions described above, a serum dilution of 1:34,000 gave 50%neutralization, which corresponded to an estimated 20 ng/ml ofTMEV-specific Abs, assuming a total serum Ig concentration of 15 mg/ml,and a TMEV-specific fraction of 5%.

Demyelination Protocol

Demyelination was induced in female SJL/J mice, ages four to six weeks,from the Jackson Laboratory, Bar Harbor, Me. Mice were inoculatedintracerebrally with 2×10⁵ plaque-forming units of DA virus in a volumeof 10 μl. Mice infected chronically with TMEV (4 to 6 months followinginfection) were assigned randomly to groups of treatment.

Treatment Protocol and Clinical Disease Assessment

Chronically infected mice were given either intraperitoneal (ip) orintravenous (iv) injections of mAb twice weekly for 4-5 weeks. At eachtreatment injection, mice were assessed clinically by three criteria:appearance, activity, and paralysis. A score for each criterion wasgiven ranging from 0 (no disease) to 3 (severe disease). For appearance,1 indicated minimal change in coat, 2 indicated a severe change(incontinence and stained coat). For activity, 1 indicated decreasedspontaneous movements (minimal ataxia), 2 indicated moderate slowing(minimal spontaneous movements), and 3 indicated severe slowing (nospontaneous movement). For paralysis, 0.5 indicated a spastic extremity,1 indicated a paralyzed extremity, 1.5 indicated two or more spasticextremities, 2 indicated two paralyzed extremities (unable to walk), 2.5indicated no righting response, and 3 indicated three or four paralyzedextremities (moribund). The total score for each mouse was thecumulative total from each criterion (maximum of 9). As the clinicalscore was an ordinal, but not a cardinal scale, the change in clinicalscore to assess clinical disease was used. The clinical assessment datawere not disclosed until after the morphological assessment ofremyelination was completed.

Light and Electron Micrograph Preparation and Assessment ofRemyelination

Preparation of light and electron microscopy sections and morphologicalassessment of remyelination were done. Briefly, treated mice wereanesthetized with pentobarbital (0.2 mg ip), exsanguinated by cardiacpuncture, and filled by intracardiac perfusing with Trump's fixative(100 mM phosphate buffer, pH 7.2, with 4% formaldehyde and 1.5%glutaraldehyde). The entire spinal cord was removed carefully from thespinal canal, and sectioned into 1 mm transverse blocks. Every thirdblock was post-fixed in 1% osmium tetroxide and embedded in Araldite(Polysciences, Warrington, Pa.). One micron sections from each blockwere cut and stained with p-phenylenediamine. On each section,remyelination was quantitated using a Zeiss interactive digital analysissystem (ZIDAS) and camera lucida attached to a Zeiss photomicroscope(Carl Zeiss Inc., Thornwood, N.Y.). Abnormally thin myelin sheathsrelative to axonal diameter was used as the criterion for CNSremyelination. Ten spinal cord sections from each mouse were examined;this corresponded to 8-9 mm² of white matter examined per mouse. Toavoid bias, slides were coded and quantitation was done withoutknowledge of the treatment groups.

Myelin Thickness and Axonal Diameter Measurements and Quantitation ofMyelination Axons

Electron micrographs of normal and remyelinated axons fromplastic-embedded spinal cord sections were imaged with a Hamamatsu videocamera, digitized, and analyzed using an IBAS 2000 Image Analysis System(Kontron, Munich, Germany). The Axonal cross-sectional area with andwithout the myelin sheath was measured, and equivalent circlecalculations were used to determine the axonal diameter and myelinsheath thickness. For myelinated axon quantitation, the number ofmyelinated axons in lesions from plastic-embedded spinal cord sectionswere counted using the analysis system described above attached to anAxiophot microscope (Carl Zeiss, Inc.). 17 remyelinated and 15demyelinated lesions in spinal cord sections from animals treated withmAb SCH94.03, control IgM, or buffer only were analyzed. Thiscorresponded to 0.6 mm² of remyelinated area and 0.8 mm² of demyelinatedarea. The criterion for selection of a lesion as demyelinated was thepresence of substantial demyelination with minimal repair, whereasremyelinated lesions were chosen based upon the presence of almostcomplete remyelination throughout the lesion.

Immunostaining

Rat 5.5B8 glial cells were grown on poly-D/L-lysine-coated chamberslides in Dulbecco's modified Eagle's medium (DMED) supplemented with1.5 g/L D-glucose, 30 nM SeO₂, 15 nM triiodothyronine, 10 ng/ml biotin,100 μM ZnCl₂ 50 μg/ml gentamicin, and 10% fetal bovine serum. Allstaining steps were done at room temperature. For surface staining,slides were briefly rinsed with PBS, and cells were lightly fixed with1% formaldehyde in PBS for 10 minutes to prevent cell detachment duringsubsequent staining steps. For cytoplasmic staining, slides were rinsedtwice in PBS and either air dried for 1 hour or incubated with 0.1%Triton X-100 in PBS for 10 minutes. Cells were blocked in 2% BSA for 30min, washed, incubated with control IgM or mAb SCH94.03 (10 μg/ml in 1%BSA) for 1 hour, and washed extensively with PBS. After fixation with 4%paraformaldehyde for 15 min, slides were incubated withfluorescein-labeled goat anti-mouse IgM (Jackson Immunoresearch) for 1hour, washed with PBS, coverslipped with 10% MOWIOL® (Hoechst) in 100 mMTris, 25% glycerol, pH 8.5 with 25 μg/ml 1,4-diazobicyclo-[2.2.2]-octane(DABCO) to prevent fading, and allowed to set overnight in the dark. Forfrozen tissue sections, fresh neonatal rat, adult mouse, or autopsiedhuman cortical brain tissue was quick frozen in isopentane chilled inliquid nitrogen prior to liquid nitrogen storage. Frozen sections (10μm) were transferred onto gelatinized glass microscope slides, air driedfor 4-8 hours, and stored at −70° C. Prior to immunostaining, slideswere placed at room temperature overnight. The immunoperoxidase stainingprotocol was similar that described above, using the ABCimmunoperoxidase reagent (Vector Laboratories, Burlingame, Calif.),developed with 1.5 mg/ml Hanker-Yates reagent (p-phenylenediamine-procatechol) in 50 mM Tris, pH 7.6 with 0.034% H202,counterstained with Mayer's hematoxylin, and mounted with Permount(Fischer Scientific, Pittsburgh, Pa.).

Data Analysis

A modified cumulative rank sum test (O'Brien, P. C., Biometrics,40:1079-1087 (1984)) was used to compare remyelination between treatmentgroups. This statistical test takes into account several numericallyunrelated parameters of therapeutic effectiveness, and is used routinelyfor clinical trial efficacy assessment. Parallel analyses using astandard unpaired Student's t-test to compare individual parameters ofremyelination gave equivalent results. Comparisons of disease severityand correlation significance were determined by a one-way analysis ofvariance (ANOVA). Statistical analyses were done with either theSigmaStat (Jandel Scientific, San Rafael, Calif.) or EXCEL (MicrosoftCorporation, Redmond, Wash.) software programs. Calculated values wereconsidered significant when p was <0.05.

Example 4

1. Hybridoma Culture and Determination of Ig Isotype

A2B5, HNK-1, and XXMEN-0E5 (anti-bacterial lipopolysaccharide)hybridomas were purchased from American Type Culture Collection(Rockville, Md.). O1 and O4 hybridomas were the gift of Dr. S. E.Pfeiffer (University of Connecticut, Farmington, Conn.). Hybridomas werecultured in RPMI 1640 containing 10% fetal calf serum (HyClone, Logan,Utah) and 2×10⁻² mM β-mercaptoethanol. IgM concentrations of thesupernatants were determined by a μ-chain-specific capture ELISA Withpurified MOPC104E (Sigma, St. Louis, Mo.) as the standard. To determinethe IgM isotype of mAbs O1, O4, and A2B5, Mouse Monoclonal AntibodyIsotyping Kit (Gibco, Grand Island, N.Y.) was used.

2. mRNA Isolation and Cloning of Ig Variable Region

Poly(A)⁺RNA was isolated from hybridoma cells by oligo(Dt)-cellulosechromatography using the Micro-Fast Track kit (Invitrogen, San Diego,Calif.). Ig heavy and light chain variable region cDNAs were cloned bythe 5′-rapid amplification of cDNA ends (RACE) method using the5′-AmpliFINDER™ RACE kit (Clontech, Palo Alto, Calif.). Briefly, firststrand cDNA was synthesized using an oligo Dt primer. An anchoroligonucleotide was ligated to the 3′ end of the first strand cDNA, andvariable region cDNAs were amplified by polymerase chain reaction usingprimers corresponding to the anchor sequence and constantregion-specific primers for the μ (CO or _(χ) (C_(χ)) chains describedpreviously [Miller et al., J. Immunol., 154 (1995), 2460-2469].

3. Sequencing and Analysis

Amplified cDNA products were purified from agarose gel afterelectrophoresis and directly subcloned into pCRII using the TA cloningkit (Invitrogen). Both strands of the insert were sequenced usingautomated DNA sequencer (Applied Biosystems model 373A, Mayo MolecularBiology Core Facility). For nucleotide sequence homology searches, theFastA program (GCG program, version 8) was used [Devereux, J. et al.,Nucleic Acids Res., 12 (1984), 387-395].

4. Direct ELISA to Determine Polyreactivity

HNK-1 was shown previously to be polyreactive by Western blots [McGarryet al., supra]. Therefore, the polyreactivity of O1, O4 and A2B5 wastested by direct ELISA. Human RBC spectrin, bovine myosin (heavy chain),mouse albumin, mouse hemoglobin, mouse transferrin, hen egg lysozyme,rabbit actin, rabbit myelin basic protein, and keyhole limpet hemocyanin(KLH) were purchased from Sigma. Proteins were tested for purity bySDS-polyacrylamide gel electrophoresis. The chemical haptentrinitrophenyl (TNP) was coupled to bovine serum albumin (BSA) [Milleret al., 1995, supra]. Protein antigens were used at 5 μg/ml, and haptenwas used at 2 μM. The proteins and hapten-BSA antigens were coated ontopolystyrene or polyvinylchloride microtiter plates in 0.1 M carbonatebuffer, pH 9.5, for 18 hours at 4° C. Coated plates were blocked withPBS containing 5% nonfat dry mild and 0.05% Tween 20 for 2 hours at roomtemperature, and incubated with mAbs diluted in blocking buffer (2μg/ml) for 4 hours at room temperature. TEPC183 (Sigma) and XXMEN-OE 5IgM mAbs were used as control antibodies. Bound IgM was detected withbiotinylated goat anti-mouse IgM (μ chain specific; JacksonImmunoresearch, West Grove, Pa.) followed by alkaline phosphataseconjugated to streptavidin, with p-nitrophenylphosphate as thechromogenic substrate. Absorbance was determined at 405 nm.

Results

Nucleotide sequences of variable region cDNA including leader peptidewere compared with published sequences of germline genes, mouse myelomaand natural autoantibodies.

1. Heavy Chain Variable Region cDNA Sequences

O1 V_(H) was identical with unrearranged V_(H) segment transcripts A1and A4 [Yancopoulos et al. Cell, 40 (1985), 271-281], which belong toV_(H)558 family (FIG. 12). The O1 D segment was relatively short andcontained four nucleotides derived from germline SP2 gene family (commonsequence to DSP2.3, 2.4 and 2.6) [Kurosawa et al., J. Exp. Med., 155(1982), 201-218]. The D segment for O1 was dG and dC rich in the 5′ end,probably representing non-coded (N) nucleotides inserted by terminaldeoxynucleotide transferase (TdT) during V-D-J recombination. O1displayed sequence identity with germline J_(H)1 [Sakano et al., Nature,286 (1980), 676-683], except for one nucleotide (GTC for GTT in thegermline), which did not result in an amino acid substitution.

Compared with the germline BALB/c V_(H)101 [Kataoka et al., J. Biol.Chem., 257 (1982), 277-285], O4 V_(H) showed three nucleotidedifferences in the V_(H) coding region (FIG. 13), all of which resultedin amino acid substitutions. Compared to natural autoantibody D23[Baccala et al., Proc. Natl. Acad. Sci. USA, 86 (1989), 4624-4628],which is encoded by germline V_(H)101, O4, V_(H) showed two nucleotidedifferences with amino acid substitutions in the V_(H) coding region.Compared with germline V_(H)101, HNK-1 V_(H) showed six nucleotidedifferences and four amino acid differences in the V_(H) coding region(FIG. 13). Compared to natural autoantibody D23, HNK-1 V_(H) showed fivenucleotide differences and three amino acid differences in the V_(H)coding region. D23 had three nucleotide differences when compared withgermline V_(H)101; all differences were also seen in the O4 and HNK-1V_(H). The O4 D segment contained five nucleotides and the HNK-1 Dsegment contained 13 nucleotides derived from germline DFL16.1 gene[Kurosawa et al., J. Exp. Med., 155 (1982), 201-218]. The HNK-1 Dsegment had one dG residue in the 5′ end and four dG residues in the 3′end, which probably represent N nucleotides inserted by TdT during V-D-Jrecombination. The heavy chain joining region of O4 corresponded togermline J_(H)4 [Sakano et al., supra]. The heavy chain joining regionof HNK-1 corresponded to germline J_(H)4 beginning with the fifth codon.

The A2B5 V_(H) showed seven nucleotide and four amino acid differencesin its coding region in comparison with the germline V1 (also called T15and S107) [Crews et al., Cell, 25 (1981), 59-66; Siu et al., J.Immunol., 138 (1987), 4466-4471] (FIG. 14). The heavy chain joiningregion of A1B5 corresponded to germline J_(H)3 beginning with the thirdcodon [Sakano et al., supra].

2. Light Chain Variable Region cDNA Sequences

Since all the hybridomas produced IgM _(χ) antibodies as determined byisotyping assay, a C_(χ) primer was used for polymerase chain reaction.O1 and O4 light chain variable region cDNA sequences were identical(FIG. 15). The V_(χ) segments of O1 and O4 were identical with naturalautoantibody E7 [Baccala et al., supra], and showed only one silentnucleotide difference when compared with myeloma MOPC21 [Hamlyn et al.,Nucleic Acids Res., 9 (1981), 4485-4494]. The J_(χ) segment of HNK-1showed sequence identity with J_(χ)2.

The genomic germline gene which encodes the V_(χ) segment of A2B5 (FIG.17) is unknown, but belongs to the V_(χ)19 group [Potter et al., Mol.Immunol., 19 (1982), 1619-1630]. The V_(χ) segment of A2B5 was identicalwith the V_(χ) segment from hybridomas H220-11, H230-2, H230-5 andH250-6 [Caton et al., J. Immunol., 147 (1991), 1675-1686] except for twonucleotide changes, one of which resulted in an amino acid substitution(data not shown). The V_(χ) segments of H220-11, H230-2, H230-5 andH250-6 are identical to each other. The J_(χ)segment of A2B5 wasidentical with J_(χ)5 [Max et al., J. Biol. Chem., 256 (1981),5116-5120; Sakano et al., supra] except for one nucleotide whichresulted in an amino acid substitution.

3. Direct ELISA

To assess the polyreactivity of the O1, O4, and A2B5, binding of mAbs toa panel of defined antigens was determined by ELISA (FIG. 18). O1reacted with human RBC spectrin. O4 reacted with human RBC spectrin,bovine myosin, mouse hemoglobin, rabbit actin, and TNP-BSA. A2B5 and thetwo control IgM_(χ)mAbs did not react with this panel of antigens.

4. Utility

The enormous diversity in the Ig variable region is due primarily tocombinations of multiple germline coding gene segments. Differentprimary structures are produced by recombination of V,D,J (heavy chain)or V,J (light chain) gene segments. Assuming the random association ofheavy and light chains to form a complete antibody molecule, the numberof different molecules is estimated to be 1.6×10⁷ [Max et al.,Fundamental Immunology, Raven Press, NY, 1993, pp. 315-382]. Somaticmutation during the process of antigen challenge provides even furtherdiversity and specificity. In contrast to the majority of Igs producedfollowing antigen challenge, natural autoantibodies are encoded directlyby germline genes with no or few to several mutations. Naturalautoantibodies are present in sera of healthy humans and rodents[Dighiero et al., J. Immunol., 131 (1983), 2267-2272; Guilbert et al.,J. Immunol., 128 (1982), 2279-2287; Hallman et al., Mol. Immunol., 26(1989), 359-370]. These natural autoantibodies are polyreactive, capableof binding to a variety of structurally unrelated antigens [Avrameas etal., Mol. Immunol., 30 (1993), 1133-1142]. The physiologic function ofnatural autoantibodies is unknown. However, by interacting with manyself constituents, these natural autoantibodies and their targets arebelieved to establish a vast network whereby the immune system canparticipate in general homeostasis.

These results provide evidence based on Ig variable region cDNAsequences that three of the four OL-reactive IgM_(χ) mAbs (O0, O4 andHNK-1) have characteristics of natural autoantibodies. The J_(H)segments of O4 and HNK-1, and the J_(χ)segments of O1, O4 and HNK-1 areencoded by unmutated germline Ig genes. The J_(χ)segment of O1 has onlyone silent nucleotide change. O1 V_(H) is identical with unrearranged VHsegment transcripts A1 and A4, which belong to the V_(H)558 family[Yancopoulos et al., supra]. Because the germline genes corresponding tothe V_(χ) genes of myeloma MOPC21 V_(χ)19 gene family [Potter et al.,supra] are unknown, direct evaluation of the somatic mutations of thelight chains was not possible. However, O1 and O4 light chain variableregions are identical with the sequence reported for naturalautoantibody E7 [Baccala et al., supra], and are identical with myelomaMOPC21 V_(χ) segment [Hamlyn et al., supra], except for one silentnucleotide change. This provides strong evidence that O1 and O4 V_(χ)segments are directly encoded by germline Ig genes. Though O4 and HNK-1V had minor differences from germline V_(H)101 [Katsoka et al, J. Biol.Chem., 257 (1982) 277-285], their sequences are very close to D23 V_(H)sequence, a well-characterized natural autoantibody [Baccala et al.,supra]. In addition, HNK-1 V_(χ)showed identity with myeloma MOPC41[Seidman et al., Nature, 280 (1979), 370-375] and germline V_(χ)41[Seidman et al., supra], except for two silent nucleotide changes. Ourresults were not able to determine whether A2B5 V_(χ) segment is encodedby germline Ig gene. However, the A2B5 V_(χ) is encoded by anunidentified germline Ig gene rather than by extensive somatic mutationof a germline Ig gene.

The results also showed that O1 and O4 react to multiple differentantigens as demonstrated by ELISA. This is consistent with theimmunocytochemistry [Eisenbarth et al., supra; Sommer et al., supra]demonstrating the reactivity of these mAbs to intracellular antigens inmany cells. HNK-1 was shown previously to be polyreactive by Westernblots using the lysates of chick embryo spinal cord neuron-enrichedcultures and rat brain [McGarry et al., supra].

The Ig cDNA sequences and polyreactivity to multiple antigens areconsistent with the hypothesis that O1, O4 and HNK-1 are naturalautoantibodies. In contrast, A2B5 does not show polyreactivity by ELISAand the Ig cDNA sequence similarity to the germline is undetermined.Characterization of O1, O4 and HNK-1 as natural autoantibodies raisesthe possibility that they exist normally in serum and have physiologicfunction during development or in CNS diseases. In support of aphysiologic function for these mAbs is the report that O4 stimulates thedifferentiation of OLs in vitro [Bansal et al., supra]. Since Schwanncells share with OLs the antigens recognized by O1, O4 and HNK-1, thissuggests that these mAbs may have a function not only in the CNS butalso in the peripheral nervous system. Direct proof of this hypothesisawaits experiments with these mAbs in vivo during development and inanimal models of CNS diseases.

Examples 5-25

The Examples that follow, present the preparation and testing of humanpolyclonal and monoclonal antibodies that correspond to the antibodiesthat correspond to the antibodies previously set forth herein. Inparticular, human polyclonal IgM antibodies were prepared, and testedwhereby their ability to bind e.g. oligodendrocytes, with a highspecificity for neural tissue, and concomitant ability to enhanceremyelination and to promote neurite outgrowth and regeneration, wasdemonstrated. As presented hereinbelow remyelination was verified in aTheiler's virus, and neurite outgrowth and regeneration have beenvalidated in the results of experiments with the well-establishedlysolecithin-induced demyelination model. Further details follow below.

Introduction

Enhancement of remyelination is an important therapeutic goal ininflammatory demyelinating disorders of the CNS such as multiplesclerosis (MS). The identification of extensively remyelinated CNSlesions in some patients dying from acute MS, and in the applicants'recent data from cerebral biopsies suggests that full repair may bepossible in the early stages of disease. However, as the diseaseprogresses, remyelination is limited and occurs primarily at theperiphery of the lesion. A number of reasons have been proposed for thefailure to achieve complete remyelination in MS lesions. Two importantconsiderations include the depletion of cells capable of remyelination,and depletion of factors, which sustain their growth anddifferentiation. Thus early intervention to stimulate reparative cellsor to remove inhibitory factors preventing myelin repair may be key to atherapeutic strategy.

A number of approaches have been tested as therapeutic strategies topromote remyelination in experimental animals. Transplantation ofoligodendrocytes or progenitor glial cells into previously demyelinatedlesions can result in remyelination of CNS axons, and to a smallerextent migration of myelinating precursor cells. It has been shown thatcentral remyelination restores conduction. An alternative strategyproposed by the applicants is to enhance endogenous remyelination. Thisapproach implies that the cells capable of remyelination and the factorswhich sustain their growth or differentiation are present indemyelinated lesions, but that there are mechanisms which inhibit thisresponse and thus prevent full remyelination.

One of the first descriptions of enhancement of endogenous CNSremyelination came from the experimental autoimmune encephalomyelitis(EAE) model. Using incomplete Freund's adjuvant (IFA) as a vehicle andmyelin components as the antigen, immunization after disease inductionpromoted structural and functional recovery in guinea pigs with EAE.Based on the promotion of endogenous remyelination in EAE, similarexperiments were conducted in mice chronically infected with Theiler'smurine encephalomyelitis virus (TMEV). TMEV infection of susceptiblestrains of mice results in chronic inflammatory demyelination in thecontext of virus persistence which serves as an excellent model of MS.Chronically infected mice treated with spinal cord homogenate (SCH) inIFA showed substantial CNS remyelination compared to control animalsgiven adjuvant alone. This observation was followed by experimentsdemonstrating that passive transfer of either antiserum or purified Igfrom uninfected syngeneic animals immunized with SCH/IFA promotesremyelination in mice chronically infected with TMEV. These experimentswere novel and contrasted the classical view that the humoral immuneresponse plays a pathogenic role in CNS demyelinating disease. Theseexperiments were the first to demonstrate that Igs, in particularautoantibodies, could play a beneficial role in promoting CNSremyelination.

Based on these observations, the generation was begun of mAbs whichpromoted CNS remyelination in the Theiler's model of demyelination.Spleens from SJL mice that had been injected with SCH/IFA were used asthe source of B cells for fusion hybridoma production. Serum from thesemice had been shown previously to promote remyelination in chronicallydemyelinated mice. Hybridomas were generated and screened by ELISA usingSCH as the antigen. After initial fusion, two of 95 viable hybridomassecreted antibodies that bound significantly to SCH. Hybridoma cellsfrom these wells (designated SCH79 and SCH94) were subcloned andscreened for SCH immunoreactivity. In the SCH79 series, 14 of 49 cloneswere positive, and for the SCH94 series 17 of 32 were positive. Themonoclonal antibodies produced by these hybridomas were then tested invivo for their ability to promote remyelination in the Theiler's modelsystem. Six to eight month chronically infected SJL mice were giveneither intraperitoneal or intravenous injection of mAbs twice weekly for4 to 5 weeks for a total dose of 0.5 mg to 5.0 mg. Two mAbs, SCH94.03and SCH94.32, showed the greatest enhancement of CNS remyelination.Sequence of variable heavy and light chains proved these two antibodiesto be identical, thus later designated as SCH94.03.

SCH94.03 treatment of SJL mice with chronic TMEV-induced demyelinationgenerally results in 20-30% remyelination of total demyelinated area, ascompared to less than 5% in PBS treated control animals. This representsa 4-6 fold increase in remyelination over controls and it is estimatedfrom axonal counts that this represents an average of 100,000remyelinated axons in SCH94.03 treated animals. Electron microscopicanalysis of fully remyelinated lesions reveals no remainingunremyelinated axons suggesting that the remyelination of availableaxons in these lesions is close to 100%.

SCH94.03 belongs to the IgM subclass and is highly polyreactive againstknown and unknown protein antigens including cytoskeletal proteins. Ofinterest, it is encoded by unmutated Ig germline genes confirming thatSCH94.03 is a natural autoantibody. Of importance, SCH94.03 recognizesan unidentified surface antigen on oligodendrocytes, providing apotential target for the mechanisms of action of this antibody.

Central to this hypothesis are the differences in the biology betweenSCH94.03 and CH12. At the time of the identification of the Ig genesequence of SCH94.03, it was discovered that there was another mouse,IgMk antibody with an identical germline sequence and gene combination.This IgM antibody, designated CH12, is from a CD5+ B cell lymphoma andhas apparent specificity to phosphatidyl choline. SCH94.03 and CH12 have99.1% amino acid identity in the V_(L) region and have identical V_(H)sequences. The only differences between SCH94.03 and CH12 are in theCDR3 region, due to N-region insertions. CH12 does not label the surfaceof oligodendrocytes and does not promote remyelination in the Theiler'smodel system, thus establishing that the only molecular difference toaccount for the mechanism of promotion of remyelination lies within theCDR3 region. This conclusion supports the hypothesis that binding ofthese mAbs to specific antigens, likely within the demyelinated lesion,is important for the induction of remyelination.

To date, six different mouse monoclonal antibodies have been identifiedwhich promote remyelination in the TMEV model for demyelinating disease.All six antibodies are of the IgM isotype and retain germline sequencesthat are reminiscent of autoantibodies. Each displays a broad antigenbinding specificity but most importantly, each binds to antigensexpressed on the surfaces of oligodendrocytes. IgM antibodies which donot bind oligodendrocytes do not promote remyelination. The prototypicmember of this group, mAb SCH94.03, has been shown to promoteremyelination in several mouse models for demyelinating disease. In micewith chronic virus-induced (TMEV) demyelination, treatment with SCH94.03results in a 4-6 fold increase in remyelination. SCH94.03 treatment hasalso been shown to significantly increase the rate of spontaneousremyelination that occurs after chemically-induced demyelinationfollowing lysolecithin injection.

Given the success with the isolation and characterization ofremyelination promoting mouse antibodies, the identification of humancounterparts to the mouse monoclonals was begun using anantigen-independent strategy. The rationale was to identify humanantibodies that react with mouse spinal cord homogenate by ELISA assay,and which bind with high affinity to structures and cells within theCNS. These antibodies were then tested in the mouse demyelination modelsfor their ability to promote myelin repair. Pooled human IgM and IgG,sera from patients from the Mayo Hematology clinic, and monoclonalantibodies from EBV-immortalized human B cells (from various sources asdescribed in the results) were characterized and tested in the mousemodels. The results of these experiments are presented below.

Results

Human Polyclonal IgM, but not IgG, Binds to Rat Brain Slices andOligodendrocytes in Culture.

By immunocytochemistry, polyclonal human IgM stains the surface of asubpopulation of oligodendrocytes (FIG. 24) and is highly reactive tostructures and cells in a slice of rat brain (FIG. 19B). No reactivityto oligodendrocyte surface antigens (FIG. 24A) or slices of rat brain,(FIG. 19A) was observed with polyclonal human IgG. Immunocytochemistryutilizing polyclonal human IgM or IgG on fixed and permeabilized mixedglial cells demonstrated minimal staining of intracellular structures(data not shown).

The specificity of polyclonal human IgM to CNS structures and cells andthe binding to oligodendrocytes may drive the pronounced remyelinatingpotential. In contrast, polyclonal human IgG, while promotingremyelination over control levels (See Table 7), does not bind to CNS,and may function by a different mechanism than polyclonal human IgM.

Human Polyclonal IgG and IgM Promote CNS Remyelination in TMEV InfectedMice.

Even though the cause of MS is unknown, epidemiologic studies suggestthat the disease may be triggered by an infectious agent (11), althoughto date no conclusive evidence proves this theory. Most recently, herpesvirus type 6 has received attention as a possible etiologic agent in asubset of patients (12). Of the multiple epidemiological factors studiedto correlate with exacerbations, only recent virus infection has beenassociated consistently (13). In addition, the major establishedtreatment for MS, IFN-β, is a cytokine central for control of virusreplication (14).

One important murine model for the study of MS is Theiler's murineencephalomyelitis virus (TMEV). This positive-stranded picornavirus hasa number of advantages: (1) The virus is a natural pathogen of mice, aspecies for which there is vast known regarding immunology and genetics;(2) Primary demyelination (destruction of myelin sheaths) is the mainphysical manifestation of chronic infection (15); (3) Host genetics playa critical role in determining susceptibility or resistance to chronicviral persistence, demyelination and neurologic disease (16-18); (4) Asin MS, pathology is immune-mediated (19-22); (5) There is completeinformation regarding the molecular virology, including details of virusreplication and virus assembly{5476,62,4100,116}. (6) The majority ofsusceptible mice harboring chronic TMEV infection develop neurologicdisease similar to MS—weakness of the extremities, spasticity,incontinence, decreased spontaneous activity and eventually paralysis(23,24).

Similar to MS, TMEV-infected mice demonstrate a wide spectrum of diseasephenotypes (defined as both demyelination and neurologic deficits) (2).At one extreme are animals in which virus replicates rapidly in CNSneurons, is not cleared by the immune system, and results in severeencephalitis and death (25). This pattern of pathology is observed inneonatal mice or mice with severe immunodeficiency. This fulminantdisease contrasts with mice that mount a protective immune response,primarily mediated by class I-restricted T cells which clear virus fromCNS. These animals develop acute encephalitis, after which virus iscleared from the CNS without subsequent demyelination (26-28). Inbetween these extremes are animals which develop and resolve acutedisease, but enter a chronic phase characterized by progressivedemyelination, and virus persistence in oligodendrocytes, astrocytes,and microglia (29,30). Although these mice mount an immune responsewhich prevents death and overwhelming encephalitis, failure to clearvirus from white matter results in chronic, persistent inflammation andimmune-mediated demyelination. To avoid anti-human immune responses,chronically infected mice (5 to 6 months after TMEV infection) weretreated with a single bolus injection of 1 mg of Ig intraperitoneally.The total dose of Ig was approximately 0.05 g/kg body weight, whichcorresponds to one eighth the total dose used for human IVIg treatment.There was no significant differences in the areas of myelin pathologybetween the treatment groups (Table 7). However, mice treated withpolyclonal human IgM demonstrated prominent remyelination. Approximatelyone quarter of the total area of myelin pathology was repaired in micetreated with polyclonal human IgM. The extent of remyelination wassignificantly higher than the spontaneous remyelination observed in thePBS-treated control groups (p<0.01), but also higher than that observedin mice treated with polyclonal human IgG (p=0.05). Mice treated withpolyclonal human IgG demonstrated more remyelination than mice treatedwith PBS. Individual lesions which were remyelinated showed almostcomplete repair with few inflammatory cells or macrophages (FIG. 30A,B).Frequently 500 to 1000 remyelinated axons were observed in each oflesion of this type. In contrast, most lesions in mice treated with PBShad few remyelinated axons and the lesions contained many inflammatorycells and macrophages, signs of active myelin destruction.

Polyclonal Human IgM Enhances Remyelination in Lysolecithin-InducedDemyelination.

Lysolecithin injection into the spinal cord is a well established methodfor chemically induced spinal cord injury and demyelination. Injectionresults in reproducible demyelinated lesions which undergo completespontaneous remyelination by 5-6 weeks. Previous work by the presentapplicants has shown that treatment with remyelination promotingantibodies increases the rate of endogenous remyelination such thatlesions are substantially repaired by 3 weeks. The monophasic nature oflysolecithin lesions, their rapid spontaneous repair, and the lack ofclinical deficits in lesioned animals, are in contrast to chronic TMEVinduced demyelination and may model aspects of spinal cord injury betterthan MS.

Animals with lysolecithin-induced spinal cord lesions were treated withpolyclonal human IgM and IgG. Photomicrgraphs of the lesion areas showedthe animals that received polyclonal human IgM contained manyremyelinated axons, while the animals treated with polyclonal human IgGor PBS contained few remyelinated axons (data not shown). In an effortto quantitate the number of remyelinated axons per area of lesion,remyelinated axons were counted under high magnification from lesions ofthe 3 treatment groups. There were significantly more myelinated axonsin lysolecithin lesions treated with polyclonal human IgM than animalstreated with polyclonal human IgG (p<0.05).

Polyclonal Human Immunoglobulins React with Multiple Self Antigens andChemical Haptens.

Antigen specificities of Igs used in this study were studied by ELISA.Previous studies have indicated the antibodies which promoteremyelination show wide reactivity to multiple protein and haptenantigens (Miller et al., 1995). Both polyclonal human IgG and IgM boundmultiple protein antigens and chemical haptens (FIGS. 33 & 34).

Polyclonal Human IgG and IgM does not React with TMEV Antigens.

To exclude the possibility that enhanced remyelination by pooled humanIgM was the result of specificity for TMEV antigens. Western blottingusing purified TMEV was performed. None of the Abs used in this studyreacted with any of the known TMEV capsid proteins (data not shown). Incontrast, rabbit polyclonal antibody raised against TMEV showed strongreactivity to the VP1, VP2, VP3 capsid antigens of the virus.

CNS-Reactive Monoclonal Antibodies can be Identified from Human Serum.

Since natural autoantibodies exist in the normal human population itshould be possible to identify human natural monoclonal autoantibodiesby screening a large number of human monoclonal IgM clones. An initialscreen was designed for polyreactivity of serum-derived human monoclonalantibodies utilizing an unfixed brain slice binding assay system.Positive clones are those samples that bound to specific brainstructures or anatomical layers or cell populations significantly abovethe background level of a fluorescently-conjugated secondary antibodyalone.

Fifty two samples of human IgM, purified from the sera of patientsobtained from the hematology department Mayo Clinic, under the directionof Dr. Robert A Kyle, were tested for CNS specificity in a brain slicebinding assay. Thirty two antibodies were determined to bind abovebackground. A variety of the reactivities are presented in FIGS. 19 and20. Fifty human serum-derived IgGs were also tested for CNS specificityin a brain slice binding assay and no distinct binding patterns abovebackground were identified (data not shown). Thus, immediately, a majordifference between human monoclonal IgMs and IgGs was identified.

The 32 positive antibodies were further tested for binding via a spinalcord homogenate ELISA system (FIG. 28) and for binding to both live andfixed rat mixed primary glial cell cultures (FIG. 25). A tabulation ofthe reactivities of those antibodies (sHIgM # antibodies) is shown inTABLE 8. The criteria for testing an antibody for biological activity invivo was CNS specificity, binding to unfixed oligodendrocytes in cultureand a significant reactivity to SCH via ELISA. Thus, sHIgM22 wasidentified as promoting remyelination. Several other sHIgM candidatesremain to be carefully studied. The ability of tested sHIgM antibodiesto bind oligodendrocytes is shown in TABLE 9.

CNS-reactive Monoclonal Antibodies were Identified from the Supernatantof EBV-immortalized Human B-cell Clones.

Supernatants from cell clones that had a total IgM concentration over 3ug/ml were tested in a brain slice assay system for their ability tobind to CNS structures. One hundred forty clone supernatants were testedfor brain slice binding. Fifteen antibodies were determined to bindabove background. A tabulation of the reactivities of these antibodies(MSI #) are shown in Table 8. A representation of ebvHIgM reactivitiesare presented in FIGS. 22 and 23. The ability of certain of theseantibodies to bind oligodendrocytes was tested. These results aretabulated in Table 9.

sHIgMs Bind to Human Cortical White Matter.

To confirm that sHIgMs bind to human CNS, human cortical white matterwas immune-labeled in system analogous to the rat brain slce assay. FIG.3 presents several of the CNS specificities of sHIgMs. Four antibodiesbind well to human CNS (FIG. 21B,C,D,E) while FIG. 21A shows a SHIgMthat binds slightly above background level.

Human Monoclonal Antibodies Bind to Surface Antigens on Cells in MixedPrimary Glial Cultures.

Several of the sHIgMs bind to cells in rat primary mixed glial cellcultures. sHIgM 12 binds to presumptive oligodendrocyte progenitorclusters (FIG. 25A) in a field of O4 positive oligodendrocytes. Fourother sHIgMs (FIG. 25B,C,D,F) bind to morphologically matureoligodendrocytes. sHIgM 30, binds to most cells (oligodendrocytes,asrocytes, microglial) in the culture (FIG. 25E).

sHIgMs and ebvHIgMs Promote CNS Remyelination in TMEV Infected Mice.

To avoid anti-human immune responses, chronically infected mice (5 to 6months after TMEV infection) were treated with a single intraperitonealbolus injection of 0.5 mg of human monoclonal antibody. Of the humanmonoclonal antibodies tested in vivo to date, sHIgM22 and ebvHIgMMSI19D10 significantly promoted remyelination over other tested humanmonoclonal IgMs (sHIgMs 1,2, and 14). There are no differences in theareas of myelin pathology between the treatment groups (Table 7). Micetreated with sHIgM22 demonstrated prominent remyelination. Approximatelyone fifth of the total area of myelin pathology was repaired in micetreated with sHIgM22 (Table 7). The extent of remyelination wassignificantly higher than the spontaneous remyelination observed inPBS-treated control groups (p<0.05, Table 7). Individual remyelinatedlesions showed almost complete repair with few inflammatory cells ormacrophages (FIG. 30). Frequently 500 to 1000 remyelinated axons wereobserved in each of lesion of this type. In contrast, most lesions inmice treated with PBS had few remyelinated axons and the lesionscontained many inflammatory cells and macrophages, signs of activemyelin destruction. Consistent with previous work demonstrating thatoligodendrocyte-reactive mouse monoclonal antibodies can promoteremyelination in vivo (Asukara et al., 1998); oligodendrocyte-reactivehuman antibodies with similar reactivities to the mouse counterparts canpromote remyelination in vivo as well.

TABLE 7 CNS Remyelination by Human Antibodies Area of CNS-Type Area ofCNS-Type Area of White Area of Myelin Remyelination RemyelinationTreatment No. of Mice Matter (mm2) Pathology (mm2) (mm2) (%) PolyclonalHuman IgG 10  8.6 ± 0.52 0.86 ± 0.10 0.13 ± 0.02 14.15 ± 2.38*Polyclonal Human IgM 13 9.70 ± 0.43 1.21 ± 0.21 0.24 ± 0.04  23.2 ±3.26** sHIgM 1 4 9.34 ± 1.93 0.68 ± 0.07 0.03 ± 0.01 8.35 ± 3.73 sHIgM 24 8.78 ± 0.70 0.87 ± 0.12 0.09 ± 0.01 11.37 ± 1.30  sHIgM 14 7 10.68 ±0.24  0.98 ± 0.09 0.08 ± 0.03 8.57 ± 2.51 sHIgM22 8 10.55 ± 0.41  1.16 ±0.22 0.19 ± 0.05 17.06 ± 3.42* sHIgM 46 5 9.44 ± 0.36 0.66 ± 0.06 0.18 ±0.04 27.12 ± 4.01  ebvHIgM MSI19D10 3 8.24 ± 0.40 0.90 ± 0.14 0.26 ±0.07  26.47 ± 3.71*** ebvHIgM AKJR4 4 8.70 ± 0.84 1.10 ± 0.15 0.05 ±0.03 4.15 ± 1.98 PBS 7 9.78 ± 0.60 1.20 ± 0.22 0.06 ± 0.02 6.74 ± 1.80Values represent the mean ± SEM. One way ANOVA and t-test were used tocompare the percent area of CNS-type remyelination in mice treated withhuman antibodies to mice treated with PBS. Such analysis revealed *p <0.05; ^(†)p < 0.01, ^(‡)p < 0.001. Comparison of mice treated withpolyclonal human IgG to other treatments revealed; polyclonal human IgMp = 0.05, sHIgM 46 P < 0.05. All other comparisons were notstatistically significant. There was no difference in the CNS-typeremyelination between polyclonal human IgM, sHIgM22 and sHIgM 46. Areaof PNS-type Schwann cell remyelination ranged from) to 0.08 mm². Thiscorresponded to 0.0 to 6.92 percent area of PNS-type Schwann cellremyelination as a function of myelinpathology. There was no statisticaldifference in the area of myelin pathology in the various treatmentgroups or compared to PBS or in the PNS-type Schwann cell remyelinationbetween groups.

TABLE 8 CLONE NAME* DESCRIPTION OF IMMUNOFLUORESCENT STAINING OFNEONATAL RAT CEREBELLUM CB2b-G8 Identified large neuronal cell bodies ingranular layer; small round cell bodies in the molecular layer; fibrousastrocytes in the central white matter. CB2e-C2 Weak label of Purkinjecell bodies; small processed cells, of oligodendrocyte and microglialmorphology, and astrocytes in central white matter and granular layer.CB2i-E12 Strong label of Purkinje cell bodies, dendritic arbors andsmall round cells in molecular layer; nearly confluent label of granularlayer; fibrous astrocytes in central white matter. CB2i-E7 labelsPurkinje cells; cells in granular layer; central white matter in thefolia; oligodendrocytes; astrocyte cytoskeleton and microglial cellsCB2i-G2 Strong fibrous astrocyte label in central white matter tracts;identifies Purkinje cell bodies; punctate label over granular layer;small cell bodies in molecular layer. CB2L-H1 Strong label of glial cellbodies in central white matter and Purkinje cell bodies; weaker label ofdendritic arbors; very pronounced small cel bodies in molecular layer.MSI 10-E10 Strong label of fibrous central white matter tracts, similarto that observed using anti-glycolipid or anti-microglial antibodies.MSI 16-E6 Strong label of Purkinje cell bodies, weaker dendritic arbors;nearly confluent label of small cells in granular layer; central whitematter nearly unlabeled. MSI 17-A2 Strong label of small round cells inmolecular layer and Purkinje cell bodies; diffuse label of granularlayer; unlabeled central white matter. MSI 19-C11 Fibrous appearance tocentral white matter with many cells of oligodendrocyte morphology;punctate surface label over most of tissue, but concentrated overpresumptive cell bodies. MSI 19-E5 Extra cellular matrix-like label ofmolecular layer; strong fibrous label of central white matter,identifying many individual glial cells. AKJR4 Identifies all neuronalcell bodies in granular layer; small round cell bodies in central whitematter and molecular layer. MSI 19D10 Binds strongly to cells of thegranular layer and to Purkinje cells and their dendritic arbors, inaddition to weakly identifying white matter and astrocytes. MSI 20H10Binds to central white matter, the granular and molecular layer andPurkinje cells with varying degrees of intensity. MSI 17E11 Binds in apunctate manner to only a few glial-appearing cells at the surface ofthe brain slice. sHIgM1 Binds the cytoskeleton of astrocytes overlyingthe central white matter of the folia. sHIgM2 Binds to cells of thegranular layer and to fibers traversing the central white matter of thefolia. sHIgM12 Binds to lend a spongy appearance to the central whitematter of the folia, and a uniform label over the molecular layer,reminiscent of an extracellular matrix molecule. sHIgM14 Binds well tocells of the granular layer and Purkinje cells located at the surface ofthe slice, while the central white matter of the folia is largely devoidof label. sHIgM22 Binds well to the cytoskeleton of damaged astrocytesoverlying the central white matter of the folia, Purkinje cells andtheir dendritic arborizations, and to small round cells in the molecularlayer, weakly labels surface of granular cells. sHIgM26 Binds tooligodendrocyte-appearing cells and fibrous white matter. sHIgM29 Bindsweakly to many structures in the cerebellum with intensity just abovebackground except for a small population of neurons in the granular andmolecular layer. Axon extensions over 100 μm are clearly delineated.sHIgM30 No binding to unfixed cerebellum sHIgM31 Binds predominantly tothe granular layer, with little binding to the white matter, Purkinjecells or astrocytes. sHIgM32 Binds to type 2 astrocyte-appearing cells.sHIgM42 Binds in a fibrous pattern to the entire folia, molecular andgranular layers and white matter. sHIgM46 Binds in a fibrous pattern tothe granular layer and white matter. The Purkinje cells are welldefined. sHIgM50 Binds predominantly to the granular layer with littlebinding to the white matter, Purkinje cells or astrocytes. sHIgM51 Bindsto small cells similar to microglia in molecular and granular layers. Ofthe 96 human EBV B-cell clones generated, 60 produced IgM antibody t aconcentration of 2 μg/ml or greater. Of these, 11 were found to stronglybind to murine cerebellum on a consistent basis. Another 10 bound tomurine cerebellum weakly or inconsistently, while 39 did not bind atall. Pictures of the immunofluorescent staining of the consistentlystrongly staining antibodies listed above as well as four representativenegative clones (CB2g-E10, CB2g-F11, CB2I-F10, MSI24-D6) areincluded.*CB: clones generated from human umbilical cord blood; MSI: clonesgenerated from the peripheral blood of multiple sclerosis patients;AKJR: clones generated from the peripheral blood of rheumatoid arthritispatients.

TABLE 9 Oligodendrocyte Binding Antibody Binding to Oligodendrocyte MSI17 A.2 negative MSI 19C11 negative MSI 19E5 labels oligodendrocyte ofmature morphology AKJR 4 negative MSI 19010 labels some oligodendrocyteof mature morphology MSI 20H10 not tested MSI 17E11 not tested sHIgM 1no reactivity to surface antigens SHIGM 2 no reactivity to surfaceantigens SHIGM 12 presumptive oligodendrocyte progenitors prior tosulfortide expression SHIGM 14 label of mature multi-processedoligodendrocyte SHIGM 22 labels mature stages of oligodendrocyte andprocess extensions SHIGM 26 no reactivity to surface antigens SHIGM 29no reactivity to surface antigens SHIGM 30 no reactivity to surfaceantigens poly hIgG negative on surface poly hIgm subset of matureoligodendrocyte CB2b G8 labels oligodendrocytes (human) of maturemorphology CB2e C2 negative CB2i E7 labels oligodendrocytes of maturemorphology CB2i E12 negative CB2i G2 negative CB2L H1 negative MSI 10E10negative MSI 16E6 negative sHIgM31 No binding to Oligodendrocyte sHIgM32No binding to Oligodendrocyte sHIgM42 Binds to mature stages ofoligodendrocyte and faintly to underlying astrocytes. sHIgM46 Stronglybinds to both mature and immature stages of oligodendrocytes withpunctate label. sHIgM50 Weak punctate label of subset of mature stagesof oligodendrocyte sHIgM51 Binds to mature stages of oligodendrocytesand faintly to underlying astrocytes.

Monoclonal Antibodies that Promote Remyelination Cause Ca²⁺ Flux inGlial Cells in Culture.

The response of cultured glial cells to physiological concentrations ofremyelination promoting antibodies suggests that these antibodies mayhave direct effects on the biochemistry of glial cells through theregulation of cellular calcium flux. This effect may represent animportant aspect of the molecular mechanism of antibody inducedremyelination. FIG. 39 demonstrates glial Ca²⁺ responses to fourdifferent antibodies. Two of these antibodies, sHIgM22 and SCH94.03,promote remyelination in vivo, and two, sHIgM 14 and CH12, do notpromote remyelination. Cells which responded to antibody, exhibited oneof two different types of calcium spikes. Some cells responded with arapid onset spike of short duration (fast response) as shown by the redtraces in panels A and B. A separate subset of cells responded with aslower onset, longer duration spike (slow response) as demonstrated bythe black traces in Panels A and B. The antibodies sHIgM22 and SCH94.03each elicited both types of responses but always from differentindividual glial cells. These qualitatively different responses clearlysuggest two distinct molecular modes of action on distinct subsets ofcells. A response to antibody (either the fast or the slow response) wasobserved in 30 of 251 cells after treatment with sHMab22 and in 36 of251 cells treated with SCH 94.03. Antibodies which do not promoteremyelination in vivo (sHIgM 14 & CH12) were not observed to causecalcium flux in cultured glia (panel C). A total of 203 cells wereexamined for each of these antibodies.

Human Monoclonal Antibodies Bind to Primary Neurons.

Many of the sHIgMs and ebvHIgMs bind to neuronal populations in brainslices. However, many of the neurons bound are at the surface of theslice, presenting the likelihood that the antibodies may bind internalepitopes inside damaged neurons. Positive binding HIgMs were tested forbinding to live rat granule cells in culture. FIG. 40 demonstrates thebinding of two sHIgMs to live neurons. sHIgM 12 binds to both axonal anddendritic extensions of the neurons (FIG. 40A), while ebvHIgm CB2iE12binds exclusively to the exterior of the granule cells membrane andproximal axon extensions (FIG. 40B). These reactivities were verified bydouble label immunocytochemistry by c-labeling human antibodies positiveneurons with anti-neurofilament and anti-microtublue associated protein2 antibodies (data not shown).

Of particular interest to our laboratory is the possibility thatdemyelination predisposes axons to immune-mediated injury andcorresponding neurologic deficits. In our recent analysis of 2microglobulin deficient mice (2 m −/−), we demonstrated that in theabsence of class I MHC, TMEV infected mice develop large demyelinatinglesions but fail to develop clinical deficits. The mice demonstrated arelative preservation of axons with increased sodium channel densitiesand remyelination of spinal cord white matter. Axonal preservationappears to be essential for the maintenance of neurologic function. Theobservation that human antibodies can bind specifically to neuronspresents another potential avenue for antibodies mediate repair of theCNS. Certain antibodies may be able to potentiate remyelination throughaction on the neuron. The repair of CNS lesions may be potentiated bymonoclonal antibodies by many possible scenerios. 1) increasing theadhesive bonds between neurons and oligodendrocytes. 2) direct cellstimulation of neuron to upregulate trophic factors and attractoligodendrocyte progenitors the area of bare axons. 3) neuroprotectionof axons by antibody blockade of leaky ion channels on bare axons. 4)protection of bare axons from recognition by activated and destructiveimmune cells.

Materials and Methods

A. Monoclonal Antibody Production, Characterization, Screening andPurification

Sources of Abs and Ab Purification.

Normal human IgM was purified from pooled plasma of over 2,500 healthydonors by modified Deutsch-Kistler-Nitschmann's ethanol fractionationprocedure followed by octanoic acid precipitation and two successiveion-exchange chromatography steps as previously described (Hurez et al.,1997). The purity of IgM was over 90% as confirmed by ELISA andSDS-polyacrylamide gel electrophoresis (PAGE). Pooled human IgG fromhealthy donors used clinically as IVIg was purchased from Miles Inc.(Elkhart, Ind.). Samples were obtained from the dysproteinase clinicunder the direction of Dr. Robert A. Kyle, Mayo Clinic. The samples ofsera came from patients with a wide variety of conditions characterizedby a monoclonal IgG or IgM spike in the serum, including Waldenstrom'smacroglobulinemia, multiple myeloma, lymphoma, benign monoclonalgammopathy.

Generation of Epstein-Barr Virus (EBV) Immortalized B Cell Lines

The B95-8 marmoset cell line was obtained from ATCC (#CRL 1612) for thegrowth and isolation of EBV. Cells are seeded at 1×10⁶ cells/ml incomplete RPMI-10 medium followed by 3 days of incubation in ahumidified, 37° C., 5% CO₂ incubator. The cells are harvested and thesupernatant is cleared by centrifugation for 10 minutes at 300×g and 4°C. The EBV-containing supernatant is passed through a 0.45 mm filter andthe flow through is collected and stored at −130° C. (liquid nitrogen).This EBV-supernatant generally contains 10²-10³ transforming units/ml.

Peripheral B cells for immortalization were collected from the blood ofnormal adults (NA), adults with rheumatiod arthritis (AKJR), adults withmultiple sclerosis (MS), and from fetal cord blood (CB). Heparinizedblood (15 ml) is diluted 1:2 in phosphate buffered saline (PBS) and 12ml of this dilution is underlayered with 12 ml of Ficoll-Hypaquein a 50ml centrifuge tube. The tube is centrifuged for 8 minutes at 1500×g, atroom temperature, and the buffy coat interface is removed andtransferred to a new 50 ml centrifuge tube. The cells are washed bycentrifugation (15 minutes, 300×g, room temperature), once in PBS andthen twice in Hank's balanced saline solution (HBSS). The cells are thenresuspended in 2-5 ml of complete RPMI-10 medium and counted.

The cells are diluted to 4×10⁶ cells/ml in complete RPMI-10 medium, 2.5ml (1×10⁷ cells) are transferred to a 50 ml centrifuge tube and 2.5 mlof EBV-supernatant is added. The tube is incubated for two hours in a37° C. water bath followed by the addition of 5 ml of complete RPMI-10medium containing 1 μg/ml cyclosporin A. The 10 ml of cell suspension isthen transferred to a 25 cm² tissue culture flask and cultured for 3weeks in a humidified, 37° C., 5% CO₂ incubator. After 3 weeks, analiquot of the culture is cryopreserved and the remainder is expandedand clonal cell lines are isolated by limiting dilution.

Purification of IgM Antibodies—

Human serum samples used for study were chosen solely by the presence ofa high IgM peak in the Ig chromatogram. Samples were obtained from thedysproteinase clinic under the direction of Dr. Robert A. Kyle, MayoClinic. The samples of sera came from patients with a wide variety ofconditions characterized by a monoclonal IgG or IgM spike in the serum,including Waldenstrom's macroglobulinemia, multiple myeloma, lymphoma,benign monoclonal gammopathy. Patients sera were dialyzed againstdeionized water during three days. Euglobulinic precipitates werecollected by centrifugation (14000 rpm/30 min.) and dissolved in PBS.Solutions were cleared by centrifugation (14000 rpm/30 min.) andchromatographed on Superose 6 column (Pharmacia, Upsalla) equilibratedwith PBS. Fractions corresponding to IgM were pooled and analyzed byreducing SDS PAGE (12% gel). IgM concentrations were determined bystaining the SDS gels with Cypro Orange (Molecular Probes, Eugene) andsubsequent scanning on Storm 840 (Molecular Dynamics). Monoclonal IgM(Sigma, St Louis) were used as a standard for concentration measuring.IgM solutions were sterilized by filtration through 0.22 μm filters.

Characterization of Antigen Binding Specificity

ELISA against mouse spinal cord homogenate (SCH) was used as an assayfor the preliminary screening of antibodies prior to in vivo testing forability to promote remyelination. To further characterize thepolyreactivity and antigenic specificity of selected antibodies, anELISA against a standard panel of protein and chemical antigens is used,as well as analysis of the antibody staining patterns in sectionedneural tissues and on cultured oligodendrocytes.

ELISA Assay

Antibodies were screened for their reactivity to mouse spinal cordhomogenate (SCH). SCH at 0.01 mg/ml coated onto polystyrene microtiterplates in 0.1 M carbonate buffer, pH 9.5, for 18 hours at 4° C., andthen washed 3× with PBS. Coated plates were blocked with phosphatebuffered saline (PBS) containing 1% BSA for 1 hour at room temperature,and then incubated with antibody diluted to 10 μg/ml in blocking bufferfor 2-24 hours at room temperature. Plates are washed three times withPBS/0.05% Tween 20 and bound antibody is then detected with biotinylatedgoat anti-IgM or IgG followed by alkaline phosphatase conjugated tostreptavidin, with p-nitrophenylphosphate as chromogenic substrate.Absorbance of the reaction is measured at 405 nm.

Antibodies are also tested for their reactivity to a panel of proteinantigens (human erythrocyte spectrin, bovine myosin heavy chain, mousehemoglobin, bovine transferrin, bovine vimentin, chicken egg lysozyme,rabbit actin, rabbit myelin basic protein, keyhole limpet hemocyanin)and bovine serum albumin (BSA)-coupled chemical haptens(4-hydroxy-3-nitrophenyl acetyl (NP), phenyloxazolone (PhoX),axophenyltrimethylammonium (TMA), fluorescein (FL),azophenylphosphoryl-choline (PC), azophenylarsonate (Ars),trinitrophenyl acetyl (TNP)). Proteins are used at 5 μg/ml andBSA-coupled haptens are used at 2 μM hapten concentration. Antigens arecoated onto polystyrene microtiter plates, reacted with antibody, andthe bound antibodies are detected as described for SCH ELISA.

Tissue Section Staining

Rat pup cerebellum is used as a source of neural tissue for thecomparison of antibody staining patterns. Fresh, unfixed, tissue isembedded in 2% low melting point agarose and cut into 300 μM saggitalsections on a McIlwain Tissue Chopper. Sections are not fixed and arekept at 4° C. or on ice throughout the rest of the procedure. Slices aretransferred into 48-well tissue culture plates in HEPES buffered Earlesbalanced salts (E/H) and blocked for 30 minutes in E/H with 5% BSA.Sections are stained with primary antibody at 10 μg/ml in E/H with 1%BSA for 2-12 hours at 4° C. Sections are washed 3× in E/H and incubatedwith an appropriate fluorescent secondary antibody in E/H with 1% BSAfor 2 hours. Sections are washed 3× in E/H, 1× in PBS and thenpost-fixed with 4% paraformaldehyde for 30 minutes. Sections are washed3× with PBS and mounted in 90% glycerin with 2.5%1,4-diazabicyclo[2.2.2]octane to prevent photobleaching.

Cultured Oligodendrocyte Staining

Cerebral hemispheres are dissected from P0-P3 Sprague-Dawley rats andthe meninges and blood vessels are removed. The tissue is minced andtransferred to a 0.25% trypsin solution in calcium and magnesium freeHEPES buffered Earles salts (E/H), 10 ml final volume per brain. Thetissue is shaken at low rpm at 37° C. for 30 minutes and then heatinactivated fetal calf serum is then added to final a finalconcentration of 10% to inactivate trypsin. MgSO₄ and DNAse I are added(to 0.1% and 20 μg/ml respectively) and the tissue is shaken for anadditional 5 minutes. The cells are washed by centrifugation andresuspended in E/H with DNase I and dissociated by trituration through aglass pipette. Large debris is allowed to settle and the overlyingcellular supernatant is washed by centrifugation through a 4% BSAcushion in E/H. The cell pellet is resuspended in culture medium and thecells are plated at 2.5×10⁵ cells per cm² on poly-D-lysine cultureplates. Plates are shaken to isolate for oligodendrocyte progenitors atday 9-12. A complete phenotypic spread of oligodendrocytes is present inthe culture at this time with progenitors present as a top layer inclusters of recently divided cells. Oligodendrocyte progenitors areisolated by gently shaking the cultures, replated on poly-lysine coatedcover slips and stimulated to differentiate by removal of growth factorsfrom the culture medium.

Live surface staining is performed at 4° C. for 15 minutes on unfixedcells after blocking with PBS and 5% BSA. Intracellular staining isperformed after fixation with 4% paraformaldehyde and permeabilizationwith 0.1% Triton X-100. Primary antibodies are detected withfluorescein-conjugated second antibodies. Coverslips are mounted in 90%glycerin with 2.5% 1,4-diazabicyclo[2.2.2]octane to preventphotobleaching and viewed on an epifluorescent microscope.

Western Blotting

Purified TMEV (Njenga et al., 1996) was separated by SDS-PAGE on 15%acrylamide gels. Proteins were transferred to a nitrocellulose membraneby electroblotting. The membrane was blocked with Tris buffered salinecontaining 5% non-fat dry milk and 0.05% Tween 20 for 2 hours at roomtemperature. The membrane was incubated with pooled human IgM, pooledhuman IgG, IgMs from two patients with Waldenstrom's macroglobulinemia,and rabbit polyclonal anti-TMEV Ab (1:2000) (Njenga et al., 1996) for 4hours at room temperature. All human Igs were used at the sameconcentration (10 ug/ml). Bound Igs were detected with biotinylated goatanti-human abs or biotinylated goat anti-rabbit abs (both from JacksonImmunoResearch Laboratories, Inc., West Grove, Pa.) and alkalinephosphatase-conjugated streptavidin using 5-bromo-4-chloro-3-indolylphosphate and nitro blue tetrazorium (BCIP/NBT):

B. Promotion of Remyelination Using Human Monoclonal Antibodies

TMEV Induced Demyelination—For the production of TMEV induceddemyelination, intracerebral virus injection is performed on 4-6 weekold animals that are lightly anesthetized with metofane. Virus isinjected using a 27 gauge needle with a Hamilton syringe that delivers a10 μl volume which contains 2×10⁵ PFU of the Daniel's strain of TMEV.Intracerebral injection results in greater than 98% incidence of chronicviral infection with demyelination. Chronically infected animals forremyelination experiments are generally 6-8 months post-infection.

Antibody Treatment Protocol

Animals with chronic demyelination receive intraperitoneal (IP)injections of purified antibodies in phosphate buffered saline. For TMEVinfected animals the injection schedule consists of twice weeklyinjections of 50 μg in 100 ml. The duration of antibody treatment isfive weeks (500 μg total dose). Animals are then sacrificed and spinalcord tissue is processed for morphological evaluation as describedbelow. For each different antibody treatment, nine chronically infected,female SJL/J mice are injected with antibody. At the end of thetreatment period, six of the animals are perfused and processed formorphometric quantitation of demyelination/remyelination and three aresacrificed for frozen tissue that is used for assessment of axonalintegrity. Three separate treatment trials, with reproducible andconsistent results, are required for any given antibody before the datais considered significant. PBS and isotype control groups are includedas negative controls for each new antibody treatment experiment.

Morphological Evaluation of Demyelination/Remyelination

At the end of each experiment the spinal cord of each animal will beassessed histologically. Mice are anesthetized with pentobarbital andperfused by intracardiac administration of fixative (phosphate buffered4% formaldehyde with 1% glutaraldehyde, pH 7.4). Spinal cords areremoved and sectioned coronally into 1 mm blocks, postfixed with osmium,and embedded in araldite. One micron-thick cross-sections are cut fromeach block and stained with 4% paraphenyldiamine. This technique isreproducible and allows consistent visualization of myelin sheaths inspinal cord white matter.

Demyelination and remyelination are quantified using a Zeiss digitalanalysis system (ZIDAS) and camera lucida (Miller and Rodriguez, 1995;Miller et al, 1994). For each mouse, ten spinal cord cross sections areexamined which span the entire cord from the cervical to the proximalcoccygeal spinal column regions. Total area of white matter, area ofdemyelination, and area of remyelination are determined for eachsection, and the areas from all ten sections analyzed for a specificmouse are added to provide total areas for each mouse. Areas ofdemyelination are characterized by large amounts of myelin debris,macrophages engulfing debris, cellular infiltration and naked axons.Oligodendrocyte remyelination is characterized by areas of axons withabnormally thin myelin sheaths and the absence of Schwann cells.Statistical comparison of the extent of demyelination and remyelinationis performed using the Student's t test.

Lysolecithin Induced Demyelination

For these experiments, 12 weeks old SJL/J mice are anesthetized withsodium pentobarbitol and a dorsal laminectomy is performed in the upperthoracic region of the spinal cord. A 34 gauge needle attached to aHamilton syringe is used to inject 1 μml of a 1% solution oflysolecithin directly into the dorsolateral aspect of the cord. Animalsare killed on day 21 post injection and the injected region of thespinal cord is removed and processed for morphological evaluation.

As a second model of demyelination, intraspinal injection oflysolecithin was used. Twelve-week-old SJL/J mice were anesthetized byintraperitoneal injection of sodium pentobarbitol (0.08 mg/g). Dorsallaminectomies were performed on the upper thoracic region of the spinalcord and lysolecithin (L-a-lysophosphatidylcholine) (Sigma, St. Louis,Mo.) was injected as described previously (Pavelko et al., 1998).Briefly, a 34 gauge needle attached to a Hamilton syringe mounted on astereotactic micromanipulator was used to inject 1% solution oflysolecithin in sterile PBS (pH 7.4) with Evan's blue added as a marker.The needle was inserted into the dorsolateral part of the spinal cord, 1ul of lysolecithin solution was injected, and then the needle was slowlywithdrawn. The wound was sutured in two layers, and mice were allowed torecover. The day of lysolecithin injection was designated day 0.

Seven days after lysolecithin injection, mice were treated with bolusintraperitoneal injection of human IgM or human IgG (1 mg/injectioneach). Control mice were treated with bolus intraperitoneal injection ofPBS. Three weeks and five weeks after the lysolecithin injection, micewere sacrificed and one (m thick sections were prepared as described inthe previous section. The araldite block showing the largestlysolecithin-induced demyelination lesion was used for quantitativeanalysis. The total area of the lesion was quantitated using a Zeissinteractive digital analysis system. The total number of remyelinatedfibers was quantitated using a Nikon microscope/computer analysissystem. The data was expressed as number of remyelinated axons/mm² oflesion.

Lysolecithin treated mice are given 50 μg IP injections of antibody ondays 0, 3, 7, 10, 14, and 17 after lysolecithin injection. Animals arekilled on day 21 after lysolecithin injection. We routinely findstatistically significant treatment effects with experimental treatmentgroups of ten animals. PBS and isotype control groups serve as negativecontrols.

C. Mechanism of Action of Remyelination Promoting Human MonoclonalAntibodies

Ca2+ Ratiometric Fluorescent Analysis

Mixed primary glial cultures, from day 2-4 postnatal rat pups, areseeded onto poly-D-lysine coated coverslips and cultured for 5-7 daysprior to analysis. Fura-2-AM and Pluronic F-127 are mixed 1:1 and addedto DMEM (serum free) to yield 4 mM Fura-2 in solution (Fura-2 loadingmedia).

Coverslips with cells are washed once with DMEM and then incubated inFura-2 loading media for 60 minutes at 37° C. The cells are then washed4 times in DMEM. The coverslip is mounted in a recording chamber on aninverted fluorescence microscope connected to a computer controlled dataacquisition system which captures digital images of 510 nm fluorescenceemission at two different wavelengths of excitation: 340 nm and 380 nm.For each recording, digital images are captured from an individual cell,at 10 second intervals over 600-800 seconds. Relative internal Ca²⁺concentration is calculated as the ratio of 340 nm/380 nm fluorescence.All recordings are made at 37° C. in 1 ml DMEM.

Test antibody is introduced by adding 50 ml of concentrated (60 mg/ml inPBS) antibody stock solution to the recording chamber to yield a finalconcentration of 3 mg/ml. After recording the effects of the addition oftest antibodies, 50 ml of a calcium ionophore stock (200 mM Br-A23187 inPBS) is added to the recording chamber to yield a final concentration of10 mM.

Discussion

Normal human immunoglobulin (Ig), especially IgG, administeredintravenously (IVIg) has been shown to be effective in treating variousautoimmune neurological diseases including Guillain-Barré syndrome (vander Mech et al., 1992), chronic idiopathic demyelinating neuropathies(van Doom et al., 1991), multifocal motor neuropathy (Chaudhry et al.,1993), polymyositis (Chemn et al., 1991), and myasthenia gravis (Edanand Landgraf, 1994). The mechanisms by which administered Ig acts isunclear. Some investigators have also suggested that this therapy may beeffective in T cell-mediated autoimmune CNS diseases such as multiplesclerosis (MS) (van Engelen et al., 1992; Achiron et al., 1992; Fazekaset al., 1997; Achiron et al., 1998; Sorensen et al., 1998).

Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinationhas been used as a model to develop novel treatments for MS. When thispicornavirus is inoculated intracerebrally in susceptible strains ofmice, TMEV induces immune-mediated progressive CNS demyelination whichis clinically and pathologically similar to MS. We showed previouslythat multiple mouse IgMκ monoclonal antibodies (mAbs) directed againstnormal CNS antigens promote CNS remyelination following TMEV-induceddemyelination (Miller et al., 1994; Asakura et al., 1998). Theprototypic antibody, designated SCH94.03, was also shown to enhance therate of spontaneous CNS remyelination following lysolecithin-induceddemyelination (Pavelko, et al., 1980) and decrease the severity andfrequency of relapses in a relapsing model of experimental autoimmuneencephalomyelitis (EAE) (31). The common features of theseremyelinating-promoting IgMκ mAbs are that they react to surfaceantigens on oligodendrocytes and have phenotypic and genotypic featuresof natural autoantibodies (Miller and Rodriguez, 1995; Asakura et al.,1998). Natural autoantibodies have wide spectrum of reactivities withself and non-self antigens. These antibodies represent a major fractionof the normal circulating IgM repertoire. Though their physiologicalfunction is unknown, the beneficial effects of natural autoantibodieshave been reported in various autoimmune disease models includingmyasthenia gravis, systemic lupus erythmatosus, and non-obese diabetes(Sundblad et al., 1989; Hentati et al., 1994; Andersson et al., 1991,1994).

IVIg is purified from human plasma pools of 3000 to 10000 healthy donorsand contains more than 95% IgG and a negligible amounts of IgM (Dalakas,1997). Based on our previous observations we hypothesized that human IgMfrom healthy donors, which is enriched in natural autoantibodies, wouldbe a more effective treatment for demyelinating disease thanconventional IVIg. To test this hypothesis we treated chronicallyTMEV-infected mice with pooled human IgM obtained from over 2,500healthy donors and examined for CNS remyelination.

In this study we demonstrated that treatment with pooled human IgM fromhealthy donors resulted in significantly enhanced remyelination byoligodendrocytes in TMEV-infected mice as compared to the treatment withpooled human IgG, or PBS. We confirmed by ELISA and immunocytochemistrythat pooled human IgM contains a population of polyreactive naturalautoantibodies to proteins and haptens. This is the first demonstrationthat polyclonal human IgM promotes CNS remyelination in models ofdemyelinating disease, thus raising the possibility that IgM fromhealthy donors may be more effective to treat human inflammatorydemyelinating diseases than conventional pooled human IgG.

To our knowledge pooled human IgM has never been tested in MS, eventhough it has been shown to be safe and effective in severe infectionsand immunodeficiency.

Natural autoantibodies are a major fraction of the IgM repertoire. Inmice natural autoantibodies are exclusively IgM, whereas in humansnatural autoantibodies are also of the IgG isotypes although with muchless frequency. To date, the only mAbs which have been shown to enhanceremyelination have been oligodendrocyte-reactive IgMκ mAbs, which havegenotypic and phenotypic features of natural autoantibodies, (Asakura etal. 1998).

In conclusion, we have demonstrated that a logical screening techniquecan be used to identify human monoclonal antibodies that have thepotential to promote remyelination in model systems of demyelination.Properties of the antibody, such as CNS specificity, the ability torecognize antigens present on oligodendrocytes and a strong binding tospinal cord homogenate, combined, can predict which antibodies are thebest candidates to test for remyelination in vivo. Many of thesemonoclonal antibodies bind well to human CNS, giving reason to hope thatsome may be useful as a therapy to successfully treat human disease.

The following is an alphabetical list of the references referred to inthis Example.

-   Asakura, K., D. J. Miller, K. Murray, R. Bansal, S. E. Pfeiffer,    and M. Rodriguez. 1996. Monoclonal autoantibody SCH94.03, which    promotes central nervous system remyelination, recognizes an antigen    on the surface of oligodendrocytes. J Neurosci Res 43:273-281.-   Asakura, K., D. J. Miller, L. R. Pease, and M. Rodriguez. 1998.    Targeting of IgMkappa antibodies to oligodendrocytes promotes CNS    remyelination. Journal of Neuroscience 18:7700-7708.-   Asakura, K., D. J. Miller, R. J. Pogulis, L. R. Pease, and M.    Rodriguez. 1996. Oligodendrocyte-reactive O1, O4, and HNK1    monoclonal antibodies are encoded by germline immunoglobulin genes.    Mol. Brain Res. 34:282-293.-   Blakemore, W. F., R. A. Eames, K. J. Smith, and W. I.    McDonald. 1977. Remyelination in the spinal cord of the cat    following intraspinal injections of lysolecithin. J. Neurol. Sci.    33:31-43.-   Crang, A. J. and W. F. Blakemore. 1991. Remyelination of    demyelinated rat axons by transplanted mouse oligodendrocytes. GLIA.    4:305-313.-   Dubois-Dalcq, M. and R. Armstrong. 1990. The cellular and molecular    events of central nervous system remyelination. Bioessays    12:569-576.-   Franklin, R. J., A. J. Crang, and W. F. Blakemore. 1991.    Transplanted type-1 astrocytes facilitate repair of demyelinating    lesions by host oligodendrocytes in adult rat spinal cord. J.    Neurocytol. 20:420-430.-   Groves, A. K., S. C. Barnett, R. J. Franklin, A. J. Crang, M.    Mayer, W. F. Blakemore, and M. Noble. 1993. Repair of demyelinated    lesions by transplantation of purified 0-2A progenitor cells Nature.    362:453-455.-   Jeffery, N. D. and W. F. Blakemore. 1995. Remyelination of mouse    spinal cord axons demyelinated by local injection of lysolecithin.    Journal of Neurocytology 24:775-781.-   Lang, W., M. Rodriguez, V. A. Lennon, and P. W. Lampert. 1984.    Demyelination and remyelination in murine viral encephalomyelitis.    Ann. N.Y. Acad. Sci. 436:98-102.-   Ludwin, S. K. 1981. Pathology of demyelination and remyelination.    Adv. Neurol. 31:123-168.-   Ludwin, S. K. 1987. Remyelination in demyelinating diseases of the    central nervous system. Crit. Rev. Neurobiol. 3:1-28.-   Ludwin, S. K. 1989. Evolving concepts and issues in remyelination.    Dev. Neurosci. 11:140-148.-   Miller, D. J. and M. Rodriguez. 1995. Spontaneous and induced    remyelination in multiple sclerosis and the Theiler's virus model of    central nervous system demyelination. [Review][119 refs]. Microscopy    Research & Technique 32:230-245.-   Miller, D. J., K. Asakura, and M. Rodriguez. 1995. Experimental    strategies to promote central nervous system remyelination in    multiple sclerosis: insights gained from the Theiler's virus model    system. J Neurosci Res. 41:291-296.-   Miller, D. J., K. S. Sanborn, J. A. Katzmann, and M.    Rodriguez. 1994. Monoclonal autoantibodies promote central nervous    system repair in an animal model of multiple sclerosis. J Neurosci    14:6230-6238.-   Miller, D. J. and M. Rodriguez. 1995. A monoclonal autoantibody that    promotes central nervous system remyelination in a model of multiple    sclerosis is a natural autoantibody encoded by germline    immunoglobulin genes. J Immunol 154:2460-2469.-   Miller, D. J., C. Rivera-Quinones, M. K. Njenga, J. Leibowitz,    and M. Rodriguez. 1995. Spontaneous CNS remyelination in beta(2)    microglobulin-deficient mice following virus-induced demyelination.    J Neurosci 1545:8345-8352.-   Miller, D. J., J. J. Bright, S. Sriram, and M. Rodriguez. 1997.    Successful treatment of established relapsing experimental    autoimmune encephalomyelitis in mice with a monclonal natural    autoantibody. Journal of Neuroimmunology 75:204-209.-   Prineas, J. W. and F. Connell. 1979. Remyelination in multiple    sclerosis. Ann. Neurol. 5:22-31.-   Prineas, J. W., R. O. Barnard, E. E. Kwon, L. R. Sharer, and E. S.    Cho. 1993. Multiple sclerosis: remyelination of nascent lesions. Ann    Neurol 33:137-151.-   Raine, C. S. and E. Wu. 1993. Multiple sclerosis: remyelination in    acute lesions. J. Neuropathol. Exp. Neurol. 52:199-204.-   Rivera-Quinones, C., D. B. McGavern, J. D. Schmelzer, S. F.    Hunter, P. A. Low, and M. Rodriguez. 1998. Absence of neurological    deficits following extensive demyelination in a class I-deficient    murine model of multiple sclerosis. Nature Med 4:187-193.-   Rodriguez, M. and B. Scheithauer. 1994. Ultrastructure of multiple    sclerosis. Ultrastruct Pathol 18:3-13.-   Rodriguez, M., E. Oleszak, and J. Leibowitz. 1987. Theiler's murine    encephalomyelitis: a model of demyelination and persistence of    virus. Crit. Rev. Immunol. 7:325-365.-   Rodriguez, M., V. A. Lennon, E. N. Benveniste, and J. E.    Merrill. 1987. Remyelination by oligodendrocytes stimulated by    antiserum to spinal cord. J. Neuropathol. Exp. Neurol. 46:84-95.-   Rodriguez, M. and V. A. Lennon. 1990. Immunoglobulins promote    remyelination in the central nervous system. Ann. Neurol. 27:12-17.-   Rodriguez, M. 1991. Immunoglobulins stimulate central nervous system    remyelination: electron microscopic and morphometric analysis of    proliferating cells. Lab Invest. 64:358-370.-   Smith, K. J., W. F. Blakemore, and W. I. McDonald. 1981. The    restoration of conduction by central remyelination. Brain.    104:383-404.-   Traugott, U., S. H. Stone, and C. S. Raine. 1982. Chronic relapsing    experimental autoimmune encephalomyelitis. treatment with    combinations of myelin components promotes clinical and structural    recovery. J. Neurol. Sci. 56:65-73.

Example 6 Screening for Epitope Mimic Peptides with an Autoantibody

In this example, the identification and preparation of peptides whichmimic the recognized antigens, or portions thereof, corresponding to theautoantibodies of the invention is described. As described earlierherein, such peptides could serve as vaccines to elicit enhanced immuneresponse to conditions indicated to be favorably responsive to increasedcirculating levels of antibodies.

An exemplary strategy for the identification of peptide mimics would beto search for peptides specifically binding, for example to the HNK-1antibody, a mouse autoantibody demonstrated to be capable of inducingremyelination. The HNK-1 epitope antigen is a carbohydrate. The HNK-1epitope is expressed predominantly on glycolipids and glycoproteins fromnervous tissue (McGarry et al., (1983) Nature 306:376-378; Ilyas et al.,(1984) Biochem. Biophys. Res. Comm. 122:1206-1211; Kruse et al., (1984)Nature 311:153-155; Yuen et al., (1997) J. Biol. Chem. 272:8924-8931).The structure which reacts with HNK-1 antibody was first described byChou and Jungalwala for the major antigenic glycolipid present in humanperipheral nerve. The composition, sugar linkage, configuration andposition of the sulfate group, were characterised as sulfate-3 GlcAβ(1-3) Galβ (1-4) GlcNAcβ (1-3) GalNAcβ (1-3) Galβ (1-4) Glcβ(1-1)-ceramide for SGGL-1 and as sulfate-3 GlcAβ (1-3) Galβ (1-4)GlcNAcβ (1-3) Galβ (1-4) GlcNAcβ (1-3) Galβ (1-4) Glcβ (1-1)-ceramidefor SGGL-2. (Chou et al, 1986).

Screening phage-displayed random peptide libraries offers a rich sourceof molecular diversity and represents a powerful means of identifyingpeptide ligands that bind a receptor molecule of interest. Phageexpressing binding peptides are selected by affinity purification withthe target of interest. This system allows a large number of phage to bescreened at one time. Since each infectious phage encodes a randomsequence expressed on its surface, a particular phage, when recoveredfrom an affinity matrix, can be amplified by another round of infection.Thus, selector molecules immobilized on a solid support can be used toselect peptides that bind to them. This procedure reveals a number ofpeptides that bind to the selector and that often display a commonconsensus amino acid sequence. Biological amplification of selectedlibrary members and sequencing allows the determination of the primarystructure of the peptide(s).

Peptide ligand identified by phage display frequently interact withnatural binding site(s) on the target molecule, and often resemble thetarget's natural ligand(s). Although this system has often been used toidentify peptide epitopes recognized by antibodies, it has also beensuccessfully used to find peptide mimics of carbohydrate molecules. Workdirected towards using peptide mimics in place of carbohydrate antigenshas been reviewed by Kieber-Emmons et al, 1998). The demonstratedability of a peptide to mimic a carbohydrate determinant indicates that,although mimicry is accomplished using amino acids in place of sugars,the specificity pattern can be reproduced.

A first screening was done with the amplified starting library of 15 merpeptides. Several clones positive in binding to the HNK-1 antibody werefound. In the initial screening with HNK-1, bound phage were eluted bypH shift, so that there was no differentiation between specifically andnon-specifically bound phage. Therefore a screening was carried outwherein HNK-1 antibody is biotinylated with a coupling agentincorporating a disulfide bridge. The biotinylated antibody ispre-reacted with the streptavidin-coated tube, unbound antibody iswashed off, and the immunotube is used for screening. Alternatively,phage are reacted with the biotinylated antibody in solution, and thenthe biotinylated complex is allowed to react with an immunotube coatedwith streptavidin. In either case, after washing away unbound phage, thebound phage are eluted by addition of dithiothreitol, which releases theantibody and the attached phage (Griffiths et al, 1994). Furthermore,these screenings were done in the presence of mouse serum (12.5%). Thisprovides a large excess of mouse IgM over the HNK-1 antibody, so thatnon-specific binding to the HNK-1 antibody should be suppressed.

In some cases, when phage in solution were allowed to react with“pre-immobilized” antibody, a rise was obtained in the number of phagebound after the third or the fourth round of selection. The clonestested bound to total mouse IgM as well. In a final experiment variousprocedures were compared in parallel: Phage were allowed to bind eitherto HNK-1 coated immunotube or to biotinylated HNK-1 in solution, and inthe presence or absence of mouse serum. An enrichment was observed usingthe pre-coated antibody, but the selected clones again bound to totalmouse IgM, although they also bound HNK-1. It is interesting to notethat the selected phage were also reactive to L2-412 antibody, whichrecognizes the same carbohydrate as HNK-1, although HNK-1 requires aterminal sulfate group, while L2-412 antibody recognizes thecarbohydrate with or without a sulfate group.

Materials and Methods

Materials

A 15-mer peptide library and E. coli K91 Kan cells may be used. The15-mer library was constructed in the vector fUSE5, a derivative of thefilamentous phage fd-tet (Scott et al, 1990). This vector carries atetracycline resistance gene allowing for selection. The filamentousphage do not kill their host; thus the infected cells becometetracycline resistant, continue to grow and secrete progeny particles.The E. coli strain K91Kan is a lambda⁻ derivative of K38 (Lyons et al,1972), has a chromosomal genotype thi and carries a kanamycin-resistancegene (mkh) (Smith et al, 1993; Yu et al, 1996). Peptides and peptide (10mg) coupled to SPDP-activated BSA (60 mg) via C-terminal cysteine, maybe obtained e.g. from ANAWA AG, 8602 Wangen, Switzerland. Tetracyclineand Kanamycin may be purchased from Sigma. L2/HNK-1 glycolipids werepurified from beef cauda equina by B. Becker in our laboratory. Sulfatedsugars, SO₃-GlcA-Gal-allyl, were kindly provided by N. Nifant'ev,Zelinsky Institutre of Organic Chemistry, Russian Academy of Sciences,Moscow.

Antibodies

Characterization and purification of the monoclonal antibody (mAbL2-412), raised in rats and recognizing the HNK-1 carbohydrate has beendescribed by Noronha, A. et al., Brain Res. 385, 237-244 (1986)). HNK-1antibody is available as TIB200 from the American Type CultureCollection (ATCC). Polyclonal rat IgG and HRP-Streptavidin were obtainedfrom Sigma (USA). HRP/anti-M13 polyclonal antibody was purchased fromPharmacia Biotech. Horseradish peroxidase (HRP)-conjugated secondaryantibody directed against rat IgG was obtained from JacksonImmunoresearch.

Amplifying the Starting Library

The primary library encoding the 15mer peptides was amplified based onthe Smith procedure (Smith et al, 1992) as follows:

The night before the cells were needed, 2 ml of LB medium (g/LBacto-Tryptone, 5 g/L NAcl, 5 g/L yeast extract), containing 100 μg/mlkanamycin, were inoculated with K91Kan cells and shaken overnight at 37°C. A 1 L flask containing 100 ml of Terrific Broth was prepared (12 gBacto-Tryptone, 24 g yeast extract, 5.04 g glycerol (4 ml) added to 900ml of water and autoclaved in 90 ml portions; 10 ml of potassiumphosphate buffer (0.17M KH₂PO₄, 0.72M K₂HPO₄, no pH adjustment required)were added to each 90 ml portion bef ore use).

The 100 ml Terrific Broth were inoculated with 1 ml of the overnightculture of K91kan cells and shaken vigorously until the OD₆₀₀ of a 1:10dilution reached 0.2. Shaking was then slowed down for 10 min to allowF-pili to regenerate and 10 μl of the starting library was added to theflask; slow shaking was continued to allow for adsorption. The culturewas then transferred to 1 L of LB containing 0.22 μg/ml tetracycline andallowed to shake vigorously for 35 minutes at 37° C. The tetracyclineconcentration was adjusted to 20 μg/ml, and an aliquot was taken fordetermination of the titer. The phage were titered (recovered titer) byplating infected cells on tetracycline medium and counting the number oftetracycline resistant colonies. An infectious unit defined in this wayis called a transforming unit (TU) and the infectivity is the ratio ofnumber of TU's to number of physical particles. Typically, an aliquot of50 μl of the culture was removed and diluted with LB containing 0.2μg/ml tetracycline (dilution range was 10³-10⁵). An aliquot of 200 μl ofeach dilution were spread on an agar-plate containing 40 μg/mltetracycline and 100 μg/ml kanamycin, incubated overnight at 37° C. Thecolonies were counted on the next day. At this stage, the titer oftetracycline resistant colonies should be about 10⁷/ml. The remainder ofthe culture was shaken vigorously overnight.

The next morning the doubly cleared supernatant obtained after 2 stepsof centrifugation (4000×g, 10 min, 4° C. and 10′500×g, GSA, 10 min, 4°C.) was precipitated overnight at 4° C. by adding 0.15 volume ofPEG/NaCl solution (16.7% polyethylene glycol in 3.3 M NaCl solution).The precipitated phages collected after centrifugation (10′500×g, GSA,40 min, 4° C.) were dissolved in 10 ml of TBS (50 mM Tris-HCl pH7.5, 150mM NaCl) and a second precipitation was carried out by adding 0.15volume of the PEG/NaCl solution to the phage suspension and incubatingfor 1 hr on ice. At this stage, a heavy precipitate should be evident.

The pellet obtained after centrifugation (14′500×g, SA600, 10 min, 4°C.) was redissolved in 10 ml TBS and transferred into a tared vesselcontaining 4.83 g CsCl. The vessel was retared and TBS was added to anet weight of 10.75 g. This should give 12 ml of a 31% w/v solution ofCsCl (density 1.30 g/ml); the solution was centrifuged 48 hrs at150′000×g at 5° C. in a SW41 rotor (Beckman). With the help of a strongvisible light source, a faint bluish non-flocculent band (containing theamplified phages) was visible above a narrow flocculent opaque whiteband (probably deriving from PEG). The phage band was collected by firstaspirating slowly the fluid overlying the phage band and then, using apipette, the phage band was withdrawn avoiding as much as possible theflocculent band underneath. The phage band was then delivered to a 26 mlpolycarbonate centrifuge bottle, which was filled to the shoulder withTBS and centrifuged in a Ti70 rotor (279′000×g, 4 h, 5° C.) andresuspended in 2 ml TBS per 1 L of culture. Phages can be stably storedin this form in a refrigerator.

The amplified library was then titered (final titer) as follows: severaldilutions of phage were prepared in TBS/gelatine (0.1 g gelatin in 100ml TBS) covering the dilution range from 10⁷ to 10¹⁰. Then 10 μl of eachof these dilutions were used to infect 10 μl of K91kan cells prepared asdescribed at the beginning of this section and each dilution mixture wasincubated 15 min at room temperature (RT) to allow phage to infect theconcentrated cells. One ml of LB containing 0.2 μg/ml tetracycline wasadded and incubated 30 min at 37° C. in a shaker-incubator. The infectedcells were then spread (200 μl) on an agar plate containing 40 μg/mltetracycline and 100 μg/ml kanamycin as described above (recoveredtiter).

Screening Procedure

A. Direct Binding

The phage library was panned using Immunotubes (Nunc., Maxisorb) coatedwith mAbL₂-412. The tubes were coated by incubating overnight at 4° C.with antibody L2-412 at 10 μg/ml protein in PBS (1 ml total volume) forthe first round and 1 μg/ml for the second and third round of screening.After blocking 2 hours with Blotto (5% non-fat dry milk, 0.05% (v/v)Tween 20 in PBS) at 4° C., 10¹¹ transforming units (in 250 μl volume) ofthe phage library per immunotube were allowed to bind 1 hour at 37° C.in a rotating chamber. For the second and third rounds, the phages werepreincubated 1 hour with 100 μg/ml of rat IgG before being added to theimmunotube, in order to decrease the number of non-specific binders.After recovery of the unbound phages (from which the negative controlphage was chosen), the tubes were washed 10 times with PBS-0.05% (v/v)Tween 20 and eluted with 0.1 M Glycine pH 2.2 (0.5-1 ml total volume),10 min. at 4° C. Eluted phages were neutralized with 1.5M Tris pH9 andthen used to infect 0.5-1 ml of log phase E. coli K91 Kan cells 15 minat room temperature. The infected bacteria were transferred to 20 ml ofLB containing 0.2 μg/ml tetracycline, and after removing an aliquot fordetermination of the titer (recovered titer), allowed to grow overnightas described in the previous section. The amplified eluate was thentwice centrifuged (10 min, 3600×g and 10 min, 14′500×g, SA600) and thefinal supernatant was precipitated with 0.15 volume of PEG/NaClovernight at 4° C. The phage was pelleted (15 min. 14′500×g, SA600) anddissolved in 1 ml PBS by pipetting and vortexing, microcentrifuged 1min. to pellet insoluble matter, and PEG-precipitated again for at least1 hr at 4° C. A heavy precipitate should be visible at this stage. Thepellet obtained after 10 min. microcentrifugation was finally dissolvedin 200 μl of PBS containing 0.02% azide. This amplified eluate can bestored and kept at 4° C. The library was subjected to three rounds ofamplification and selection.

The same procedure was used for the HNK-1 screening with HNK-1 antibody,except that a 100-fold excess of mouse lgM was included to decreasenon-specific binding.

The phage were titered (final titer) as described. The colonies werecounted on the next day and the yield of the screening was calculated bydividing the recovered titer by the titer (input) of the previous round.

B. Screening with Biotinylated Antibody

Two procedures were used to accomplish this screening, both followingprotocols of G. Smith (unpublished protocols). The HNK-1 antibody wasbiotinylated as described below using NHS-SS-biotin. NHS-SS-Biotin linksthe biotin to the protein via a disulfide bridge, in order to allow thebiotin group to be subsequently removed by incubation withdithiothreitol (DTT). The L2-412 antibody was similarly biotinylated asdescribed below. In procedure A, the biotinylated antibody is firstallowed to bind to a streptavidin coated immunotube, which is thensubsequently used to pan the phage input. In procedure B, thebiotinylated antibody is preincubated with the phage in solution, andthe reaction mixture is allowed to bind (a few minutes) to thestreptavidin-coated immunotube.

In procedure A, the immunotubes were coated with 10 μg/ml streptavidinin PBS, 1 ml total volume (wet the entire surface of the tube),overnight at 4° C. on a rotator. Streptavidin was discarded and the tubewas filled with blocking solution, PBS containing 0.5% (w/v) BSA, for 2hrs at 4° C. After washing 6 times with PBS-0.05% (v/v) Tween 20(PBS-T), the biotinylated antibody was added. Typically, 3 μg of thebiotinylated HNK-1, or 5 μg of the biotinylatedL2-412 antibody wereadded in 400 μl of the blocking solution. The antibody was allowed tobind for at least 2 hrs (or overnight) at 4° C. on the rotator. Afterwashing 6 times with PBS-T, 10¹⁰ phages from the 15-mer startinglibrary, in 400 μl of blocking solution, were allowed to bind to therespective antibody-coated immunotube for 4 hr at 4° C. on the rotator.In procedure B, during coating of the immunotubes 10¹⁰ phage werepreincubated overnight with 3 or 5 μg of the biotinylated HNK-1 orL2-412 antibody, respectively. The biotinylated antibody was thenallowed to bind to the coated immunotube for 10 minutes at 4° C. on therotator. In both procedures, the tubes were then washed 10 times, thenphage-antibody complexes were eluted with 20 mM DTT (0.5 ml volume) inPBS 1-5 min. at room temperature. Amplification and titering wereperformed as described above. The library was subjected to four roundsof amplification and selection.

ELISA Screening

A. Direct Binding for Detection of Positive Clones

Individual colonies resistant to tetracycline and kanamycin were grownin LB containing 20 μg/ml tetracycline in 96-wells plates (Nunc)overnight at 37° C. (300 μl/well), then centrifuged 10 minutes at 3000rpm in Jouan centrifuge and the supernatant (100 μl) was incubated for 2hr in another 96-well plate previously coated with mAb_(L2)-412 (100 μl,μg/ml overnight at 4° C.) and blocked by incubation for 2 hours withPBS-0.5% (w/v) BSA. After washing 5 times, the binding of the phages wasdetected by incubation with HRP-conjugated anti-M13 antibody (Pharmacia,Biotech.) for 1 hour at a dilution of 1:2000. The peroxidase reactionwas started by the addition of 100 μl developer containing 0.01%hydrogen peroxide and 0.1% (w/v)2,2T-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)-diammonium salt(ABTS, Boehringer Mannheim) in HRP buffer (0.1 M sodium acetate, 0.05MNaH₂PO₄, pH adjusted to 4.2 with acetic acid). The absorbance of thecolored reaction product was determined at 405 nm in a MultiscanTitertekPlus (Flow, Switzerland). In parallel, each clone was alsotested on 96-well plates coated with rat IgG, (100 μl, 1 μg/ml in PBSand identically blocked for 2 hours). Bacteria producing the selectedbinding clones (named positive phage), that were positive binders forthe mAb_(L2)-412 but did not bind to rat IgG were streaked on an agarplate containing LB medium with 40 μg/ml tetracycline and 100 μg/mlkanamycin. Two individual colonies were picked and re-assayed forpositivity towards mAb L2-412. Positive single colonies were stored in40% glycerol at −80° C.

B. Competition Binding

Microtiter plates (Nunc) were coated with the L2/HNK-1 glycolipids (50μl, 1 μg/ml, dissolved in EtOH) and allowed to dry overnight. Whileblocking the wells for 2 hours with 0.5% (w/v) fatty-acid-free BSA inPBS, a limiting concentration of_(L2)-412, previously determined, waspre-incubated with successive 2-fold dilutions of the inhibitor,starting at a concentration of 2.2 mM for the free peptide, 5 mM for theSO₃ sugar and 10¹² positive and negative phages (the negative phageswere cloned from the unbound fraction of the first round of screening).The pre-incubated mixture was then added to the well in 100 μl andincubated for 1 hour at RT. After washing 5 times with PBS-0/05% (v/v)Tween 20, the binding of mAb L2-412 was detected by incubation withHRP-conjugated goat anti-rat IgG for 1 hour, followed by the colorreaction described earlier. The percentage of inhibition of the bindingof mAb L2-412 to the substrate in the presence of the inhibitor wascalculated with reference to the control value obtained in the absenceof inhibitor (0% of inhibition).

C. Inhibition of Binding

Microtiterplates were coated overnight at 4° C. with laminin(Gibco/BRL), (10 μg/ml, 100 μl), or mAb_(L2)-412 (1 μg/ml, 100 μl) inPBS. All the following reaction steps were carried out at roomtemperature. After blocking with PBS+0.5% (w/v) BSA, 50 μl of successive2-fold dilutions of peptide coupled to BSA (ANAWA Ag, Switzerland)starting at a concentration of 30 μM was added for 1-2 hours at RT. Thena limiting number of phages bearing the peptide of interest, previouslydetermined, was added and incubated for another hour. The bound phageswere detected with HRP/anti-M13 antibody as described in the ELISAscreening section. The analogous experiment was done with immobilizedL2-412 instead of laminin, the peptide coupled to BSA competing with thebinding of positive phages to the antibody L2-412.

D. Direct Binding to Laminin

Microtiter plates were coated with 100 μl of mAb L2-412 or laminin asdescribed above and 100 μl of biotinylated peptide coupled to BSA wasadded starting at a concentration of 30 μM, incubated 2 hours at roomtemperature, and detected with HRP-streptavidin.

DNA Sequencing

Positive clones, toothpicked from frozen glycerol stocks, were grownovernight at 37° C. in LB containing 20 μg/ml tetracycline. Singlestranded DNA was purified as described by G. Smith (1992) using thedouble-spin method, sequenced with the Thermo Sequenase cycle sequencingkit (Amersham), and loaded on an automated sequencer (B10 GeneticAnalyzer, Applied Biosystems Inc.).

Biotinylation

Biotinylation of the HNK-1 antibody, BSA and the peptide coupled to BSAwas done using Sulfo-NHS-biotin (Pierce) according to the manufacturer'sinstructions. A molar ratio of 10 to 1 was used for the antibody and 5to 1 for BSA or the peptides coupled to BSA. The biotinylated productwas dialysed overnight against PBS at 4° C.

Neurite Outgrowth Experiments and Culture

Preparation of Motor Neurons

Cover slips were sterilized by baking them overnight at 160° C. andcoated by an overnight incubation with polyornithine (Sigma, 1.5 μg/mlin water) at 4° C. The cover slips were then washed 3 times with waterand further coated with test substances as follows: 1) The BSA-peptideconjugates were dissolved at 100 μg/ml in PBS, sonicated 1 min with atable sonicator and centrifuges in a microfuge for 20 min at maximumspeed. The protein concentration of the supernatant was determined eachtime by the method of Bradford (Bradford et al, 1976). Then 120 μlcomplex was mixed with 280 μl of collagen solution (20 μg/ml collagen inPBS) and 100 μl were applied on each cover slip overnight at 4° C.; 2)As a negative control, untreated BSA was used in place of thepeptide-BSA complex; 3) The glycolipids carrying the L2/HNK-1carbohydrate were dissolved in ethanol at a concentration of 10 μg/ml,and 80 μl were added to 1 ml of the collagen solution described above. Avolume of 100 μl was used for coating. Cover slips were placed inquadruplicate in a 24-well plate (NUNC), and finally washed 3 timesbefore the cells were plated (the cover slips were never allowed todry).

Motor neuronal cells were prepared as described by Arakawa (1990) fromspinal cord of 6-day old chick embryos dissociated in 1 ml of ice coldsolution containing 0.05% DNAse 1 (Sigma), 0.1% BSA in L-15 medium (LifeTechnologies). Cells were layered on 2 ml of 6.8% Metrizamide (Fluka) inL-15 and centrifuged 15 minutes at 500×g, 4° C. Cells collected from theMetrizamide/medium interface were diluted in 5 ml L-15 and loaded on a 4ml cushion of BSA (4% BSA in L-15) and centrifuged 10 minutes at 300×g,4° C. The pellet was resuspended in 0.5-1 ml of complete medium ((22 mMNaHCO₃, 22 mM glucose, 1% of penicillin and streptomycin (Gibco) in L-15supplemented with 1% N2 supplement (Gibco) and 15 μg/ml chicken muscleextract (3.5 mg/ml). 30,000 cells were plated on poly-ornithine/collagencoated cover slips in the presence or absence of the peptide coupled toBSA and incubated in a humidified chamber at 37° C. and 5% CO₂. Thelength and number of neurites were measured and counted for isolatedneurons that were not in contact with other cells and with at least oneprocess that was as long as the diameter of the cell body after 24 hoursof culture.

Preparation and Culture of Dorsal Root Ganglion Neurons

The cover slips were prepared identically as for the experiments withmotor neurons. Dorsal root ganglia neurons were isolated fromembryonic-day 11 chicken eggs. The ganglia were transferred into 1 ml ofdigestion solution (0.05% Trypsin, 0.01% DNAse 1 in HBSS medium) andincubated 15 min. at 37° C. with resuspending every 2-5 min. The gangliawere then dissociated in 1 ml of ice cold dissociation solution (0.05%DNAse 1, 0.1% BSA, in L15 medium), loaded on 3 ml of a 4% BSA cushion ina 15 ml Falcon tube and centrifuged at 4° C., 600×g for 20 min. Thecells were resuspended in 0.5 ml of the complete medium described in theprevious section. 20,000 cells were added to wells containing one coverslip, and allowed to grow for 18 hrs in a humidified chamber at 37° C.and 5% CO₂. Fixing and analysis of neurite outgrowth was performed asdescribed in the preceding section.

Immunohistology and Immunocytology

Immunohistology

Cryosections of femoral nerve from a 4-month-old mouse were used to lookfor binding of peptide-BSA complex. The sections were treated for 1 hrwith 1% H₂O₂, 0.5% bovine serum albumin (BSA), and 10% goat serum inPBS, in order to reduce the endogenous peroxidase activity. The sectionswere then incubated overnight at 4° C. with peptide-BSA complex or BSA(1 mg/ml in PBS, 150 μl/cover slips), and then washed 4 times withPBS-0.01% Tween 20. For detection, anti-BSA antibody (Sigma, 1:16dilution, 150 μl/cover slips) was added and incubated overnight at 4° C.HRP-coupled goat anti rabbit serum was added (1:2000), for 1 hr in avolume of 150 μl per cover slip. The color reaction was developed usinga 5% dilution of a 4 mg/ml stock solution of 9-amino-3-ethylcarbazol(AEC, Fluka) in N,N′-dimethylformamide in 0.1 M sodium acetate buffer,pH 4.8, containing 0.1% H₂O₂.L2-412 antibody and HRP-coupled goatanti-rat antibody were used for the positive control. A similarexperiment was performed using biotinylated BSA-peptide conjugate. Aconcentration of 50 μg/ml was used for the overnight incubation andHRP-coupled streptavidin (1:2000) was added for 1 hr. The color reactionwas developed as described above.

Immunocytology

Cover slips were coated with polyornithine (1.5 μg/ml) then withcollagen (20 μg/ml,) and 40,000 cells were allowed to grow for 40 hrs at37° C. under 5% CO₂ as described above. The fixed cover slips were thenblocked in 5% non-fat dry milk powder in PBS for 2 hrs. After extensivewashing with PBS-0.05% Tween-20, biotinylated BSA-peptide conjugate wasadded at a concentration of 50 μg/ml for 4 hrs. After another six timeswash steps, detection was done using HRP-coupled streptavidin, 1:500,for 1 hr. Color detection was as described above for immunohistology.The fixed neurons were photographed at 40× magnification. The imagespresented were processed for enhanced color rendition using AdobePhotoshop.

The following is an alphabetical list of the references referred to inthis Example.

-   Chou, D., and Jungalwala, F. J. Biol. Chem. 268, 21727-21733 (1993).-   Chou, D. K., et al., J. Biol. Chem. 261, 11717-25 (1986).-   Griffiths, A. et al. (1994) EMBO J. 13:3245-3260.-   Kieber-Emmons, T. Immunologic Research 17, 95-108 (1998).-   Lyons, L. and Zinder, N. (1972) Virology 49:45-60.-   Scott, J. K. and Smith, G. P. (1990) Science 249:386-390.-   Smith G. P. and Smith, J. K. (1993) Methods Enzymol. 217:228-257.-   Yu, J. and Smith, G. (1996) Methods Enzymol. 267:3-27.

Example 7 Human Monoclonal Antibodies Reactive to OligodendrocytesPromote Remyelination in a Model of Multiple Sclerosis

Promoting remyelination, a major goal of an effective treatment fordemyelinating diseases, has the potential to protect vulnerable axons,increase conduction velocity and improve neurologic deficits. Strategiesto promote remyelination have focused on transplanting oligodendrocytes(OLs) or recruiting endogenous myelinating cells with trophic factors.Immunoglobulin (Ig) based therapies, routinely used to treat a varietyof neurological and autoimmune diseases, underlies our approach toenhance remyelination. We isolated two human monoclonal antibodies(mAbs) directed against OL surface antigens that promoted significantremyelination in a virus-mediated model of multiple sclerosis (MS). Fouradditional OL-binding human mAbs did not promote remyelination. Bothhuman mAbs were as effective as human intravenous immunoglobulin (IVIg),a treatment shown to have efficacy in MS, and bound to the surface ofhuman OLs suggesting a direct effect of the mAbs on the cellsresponsible for myelination. Alternatively, targeting human mAbs toareas of central nervous system (CNS) pathology may facilitate theopsonization of myelin debris allowing repair to proceed. Human mAbswere isolated from the sera of individuals with a form of monoclonalgammopathy. These individuals carry a high level of monoclonal proteinin their blood without detriment, lending support to the belief thatadministration of these mAbs as a therapy would be safe. Our resultsare 1) consistent with the hypothesis that CNS-reactive mAbs, part ofthe normal Ig repertoire in humans, may help repair and protect the CNSfrom pathogenic immune injury and 2) further challenge the premise thatAbs that bind OLs are necessarily pathogenic.

Introduction

Enhancement of remyelination and protection from axonal injury areimportant therapeutic goals in the treatment of inflammatorydemyelinating CNS disorders such as MS. Remyelination in MS plaques canoccur, but is limited (1,2) even though OL progenitors are present inthe adult (3,4). A number of therapeutic strategies to promoteremyelination have been tested in experimental animals. Transplantationof OLs (5) or their progenitors (6) into demyelinated tissue producesnew myelin. Transplanted OL progenitors can also remyelinatedemyelinated lesions in the adult CNS (7) and migrate toward an area ofdamage when placed in close proximity to the lesion (8). Unresolvedissues remain concerning the survival of transplanted OL progenitors inthe intact adult CNS and their ability to target to areas of myelinpathology (9). However, if CNS lesions are surgically approachable andaxons are still intact, transplantation of glial cells maybe a viabletherapy for improving functional performance (10).

The in vitro administration of growth or trophic factors induces theexpansion of OL progenitors (11,12) or promotes mature OLs todedifferentiate and subsequently reinitiate a program of myelination(13,14). The in vivo administration of trophic factors via geneticallyengineered fibroblasts to the injured CNS promotes axonal sprouting andOL proliferation (15). Obstacles to in vivo trophic factor therapyremain, specifically determining the biologically relevant local factorconcentration and the potential pleiotropic roles of most trophicfactors administered in high concentrations.

As an alternative, our laboratory proposes to repair CNS pathology andenhance endogenous remyelination by using CNS-binding Igs (16), buildingon a natural reparative response that may already be upregulatedfollowing demyelination. Ig therapy can be rapidly adapted and tested asa treatment for human demyelinating disease (17, 18). The premise of ourapproach is that cells capable of remyelination—and the factorsnecessary to sustain their growth and differentiation—are present in thedemyelinated CNS, but their capacity to produce myelin is limited. Theemerging heterogeneity of pathology and OL sparing within the MSpopulation (19) suggests that in practice, the treatment of humandemyelinating disease may require combinations of several therapeuticapproaches based on an individual's requirements.

We have used a virus-mediated model of demyelination to develop Ig-basedtherapy. When Theiler's murine encephalomyelitis virus (TMEV) isinoculated intracerebrally into susceptible strains of mice, TMEVinduces immune-mediated progressive CNS demyelination clinically andpathologically similar to MS (20). The efficacy of therapies in human MSclosely parallel those observed in the TMEV model (21) making this animportant platform for the design of clinical trials. A mouse mAb raisedagainst spinal cord homogenate, designated SCH94.03, enhancesremyelination in the TMEV model (22). SCH94.03 is a polyreactive, mouseIgMk mAb that binds to the surface of OLs (23). SCH94.03 also enhancesthe rate of spontaneous CNS remyelination following lysolecithin-induceddemyelination (24) and decreases relapse in experimental autoimmuneencephalomyelitis (EAE) (25). Additional OL-binding mouse IgMk mAbs,several of which are routine markers for the OL lineage, also promoteCNS remyelination (26).

Since mouse IgM mAbs promote remyelination, we hypothesized thatpolyclonal human IgM would be a more effective treatment ofdemyelinating disease than IVIg, an established therapy forimmune-mediated disorders (27). Treatment of chronically TMEV-infectedmice with polyclonal human IgM resulted in enhanced remyelination whencompared to IVIg. Two human IgM mAbs were also identified, using anantigen-independent strategy, which promote remyelination to anequivalent or greater degree than polyclonal human IgM. We suggest thathuman remyelination-promoting mAbs may be an easily implemented,effective therapy for human demyelinating disease. Human mAbs arereadily applicable to clinical trials, can be produced free ofinfectious agents and may alleviate the national shortage and high costof IVIg. An effective human mAb that promotes remyelination may alsosimplify the investigation for the mechanism of action ofimmunomodulatory therapies.

Materials and Methods

Human Antibodies and Their Isolation

Normal human IgM purified from the pooled plasma of more than 2500healthy donors was obtained from S. V. Kaveri (28). The purity of IgMwas more than 90% as confirmed by SDS-PAGE. Pooled human IgG fromhealthy donors designated clinically as IVIg was from Miles Inc(Elkhart, Ind.).

Human serum samples were obtained from the dysproteinemia clinic underthe direction of Dr. Robert A. Kyle, Mayo Clinic, and chosen solely bythe presence of an Ig clonal peak of greater than 20 mg/ml. Sera werefrom 102 patients with a wide variety of conditions characterized by amonoclonal IgG or IgM spike in the serum, including Waldenstrom'smacroglobulinemia, multiple myeloma, lymphoma, and monoclonal gammopathyof undetermined significance. Sera were dialyzed against water, theprecipitates collected by centrifugation (14,000 rpm/30 min) anddissolved in PBS. Solutions were centrifuged and chromatographed onSuperose-6 column (Pharmacia, Upsalla, Sweden). IgM fractions werepooled and analyzed by SDS PAGE. Concentrations were determined by gelstaining with Sypro Orange (Molecular Probes, Eugene, Oreg.)densitometry. IgM solutions were sterile filtered and cryopreserved.

OL Cell Culture and Immunocytochemistry

Cerebral hemispheres from P0-P2 Holtzman Sprague-Dawley rats wereprepared for mixed primary glial cell culture as described (29) andgrown for 9 days in vitro. Rat OL progenitors were isolated as described(30). Adult human OLs were prepared from temporal lobe biopsies obtainedfrom patients undergoing therapeutic resection for intractable epilepsy.Tissue did not contain the epileptic focus and was of normalcytoarchitecture when examined by the Department of Surgical Pathology.Adult glial cells isolated as described (31) and seeded ontopoly-ornithine (Sigma) and laminin (Life Technologies) coated plasticmulti-wells (Becton Dickenson) or glass coverslips (Fisher Scientific)in a defined media of DMEM/F12 supplemented with biotin (0.01 mg/ml),tri-iodotyronine (15 nM), 0.5% BSA (all from Sigma), N2, 1% pen/strep(both from Life Technologies) and recombinant human PDGF AA ® & DSystems, Minneapolis, Minn.). Cell surface staining was done at 4° C.for 12 min on unfixed cells after blocking with HEPES-buffered EBSS(E/H) with 5% BSA. All human Abs were used at 10 mg/ml. Intracellularstaining for myelin basic protein using polyclonal mouse antisera(Boehringer Mannheim) was done at room temperature after fixation with4% paraformaldehyde and permeabilization for 5 min with 0.05% saponin.Primary Abs were detected using fluorescently-conjugated secondary Abs(Jackson ImmunoResearch Laboratories, West Grove, Pa.). Cell monolayerswere mounted in 90% glycerin/PBS with 2.5% 1,4-diazabicyclo[2.2.2]octaneto prevent fading (37) and 0.1 μg/ml bisbenzimide (both from Sigma) andviewed with an Olympus Provis epifluorescent microscope equipped with aSPOT digital camera (Diagnostic Instruments Inc, Sterling Heights,Mich.).

Virus and Animals

The Daniel's strain of TMEV was used for these experiments and wasprepared as described (32). Female SJL/J mice from the JacksonLaboratories were used after 1-week acclimation. Mice 4- to 6-weeks ofage were injected intracerebrally with 2×105 plaque forming units ofTMEV in 10 ml volume resulting in greater than 98% incidence of chronicviral infection. Animals used in this study were 5 to 8 monthspost-infection and received a single intraperitoneal injection of Ig orPBS. Dosages were 1.0 mg of IVIg or human polyclonal IgM or 0.5 mg ofthe human mAbs. Animals were killed 5 weeks following Ab treatment formorphologic assessment; chosen because studies in toxic models ofdemyelination indicate that CNS remyelination is almost complete by thistime (33). Spinal cord sections embedded in plastic were cut by acentralized microscopy facility and returned to the laboratory markedwith a numerical code. In this way slides are graded for remyelinationin a blinded manner.

Western Blotting

Purified TMEV (34) was separated by SDS-PAGE and proteins transferred tonitrocellulose. After blocking with Tris buffered saline containing 5%non-fat dry milk and 0.05% Tween 20 for 2 hours at room temperature themembrane was incubated with human Igs (10 μg/ml) or rabbit polyclonalanti-TMEV Ab (1:2000) for 4 hours. Bound Igs were detected withbiotinylated goat anti-human mAbs or biotinylated goat anti-rabbit mAbs(both from Jackson ImmunoResearch) and alkaline phosphatase-conjugatedstreptavidin using 5-bromo-4-chloro-3-indolyl phosphate and nitro bluetetrazorium (BCIP/NBT, KPL, Gaithersburg, Md.).

Quantitation of Spinal Cord Demyelination/Remyelination

We have developed methods to quantify the amount of spinal corddemyelination, remyelination and atrophy in susceptible mice usingplastic-embedded cross sections stained with 4% paraphenylenediamine(PPD) to visualize myelin (35, FIG. 42A). To obtain a representativesampling of the entire spinal cord, 1 mm thick cross sections were cutfrom every third serial 1 mm block, generating 10 to 12 cross sectionsthat represent the whole spinal cord. From each cross section the areaof white matter, white matter pathology, OL remyelination, and Schwanncell (SC) remyelination were calculated using a Zeiss interactivedigital analysis system (ZIDAS) and camera lucida attached to a Zeissphotomicroscope (Carl Zeiss Inc., Thornwood, N.Y.). White matter wasoutlined at a magnification of 40×. The areas of white matter pathology,defined as regions of white matter with demyelination or remyelination,were then traced at a magnification of 100×. Regions of white matterpathology often contained macrophage infiltration, inflammation, andlittle or no PPD stain (FIG. 43 C, D, H). The sum of the areas ofpathology containing primary demyelination with or without remyelinationwas determined as a measure of total demyelination.

The areas of remyelination, either OL or SC, were traced at amagnification of 250×. OLs can remyelinate multiple axon fibers, andthus, OL remyelination results in densely packed, yet thin, myelinsheaths compared to spared normally myelinated axons. SCs canremyelinate only a single axon fiber, resulting in thicker myelinsheaths and increased space between axon fibers compared OLremyelination. SC bodies and nuclei can be observed adjacent to theaxons they have remyelinated. Total areas were calculated for each mouseby summing all the areas traced from each of 10 to 12 spinal cordsections per mouse.

The percent area of spinal cord white matter pathology per mouse wasobtained by dividing the total area of white matter pathology by thetotal area of white matter sampled. The percent area of remyelinationper mouse was obtained by dividing the area of OL or SC remyelination bythe total area of white matter pathology. Repeated measures of whitematter pathology and extensive myelin repair revealed comparable valuesdiffering only by 1.5%. To determine the validity of using 10 crosssections as a representation of the remyelination throughout the spinalcord, a comparison was performed using 10 cross sections versus all 32cross sections of a single chronically infected mouse. Assaying 10 crosssections resulted in a percent area remyelination value of 47.7%,whereas the data from all 32 cross sections resulted in a value of40.0%. Either value would have indicated significant remyelination inour assay.

Results

Human IVIg and Polyclonal Human IgM Promote CNS Remyelination inTMEV-Infected Mice

Clinical studies in MS indicate that IVIg may be partially effective instabilizing the disease course (18,36,37). To determine if human IVIgcould promote remyelination in the TMEV model of MS, chronicallyinfected mice were treated with a single intraperitoneal injection of 1mg of IVIg. A single dose was administered to avoid evoking an immuneresponse to the foreign Ig. The total dose of human Ig was approximately0.05 g/kg body weight, one-quarter the total dose used for human IVIgtreatment (18). Additional mice were treated with a single 1 mg bolus ofpolyclonal human IgM. Upon examination of the spinal cords, the percentarea of OL remyelination in mice receiving either IVIg or polyclonalhuman IgM (Table 10, 14.15% and 23.19%, respectively) was significantlyhigher than the spontaneous OL remyelination observed in the PBS-treatedgroup (6.74%, p<0.05 for IgG, p<0.01 for IgM). There were nostatistically significant differences in the areas of white matter orthe areas of white matter pathology between either treatment group orthe PBS control group. The data describes two independent experimentstreating groups of 7 and 9 mice with IVIg and groups of 7 and 10 micetreated with polyclonal human IgM. The final values in Table 10 includeonly those animals that contained at least 5% white matter pathology.

TABLE 10 CNS remyelination in mice after treatment with human Abs Areaof CNS No. type Area of CNS- of Area of white Area of myelinremyelination, type Treatment Mice matter, mm² pathology, mm² mm²remyelination, % IVIg 10 8.60 ± 0.52 0.86 ± 0.10 0.13 ± 0.02 14.15 ±2.38* Human 14 9.70 ± 0.43 1.21 ± 0.21 0.24 ± 0.04 23.19 ± 3.26† IgmsHIgM 1 4 9.34 ± 1.93 0.68 ± 0.07 0.03 ± 0.01 8.35 ± 3.73 sHIgM 2 4 8.78± 0.70 0.87 ± 0.12 0.10 ± 0.01 11.37 ± 1.30  sHIgM 14 7 11.01 ± 0.60 1.13 ± 0.18 0.08 ± 0.03 8.41 ± 2.59 sHIgM22 8 10.55 ± 0.41  1.16 ± 0.220.19 ± 0.05 17.06 ± 3.42* sHIgM 46 5 9.44 ± 0.36 0.66 ± 0.06 0.18 ± 0.0427.12 ± 4.01‡ PBS 7 9.78 ± 0.60 1.20 ± 0.22 0.06 ± 0.02 6.74 ± 1.80Values represent the mean ± SEM. One-way ANOVA and t test were used tocompare the percent area of CNS-type remyelination in mice treated withhuman antibodies to mice treated with PBS. Such analysis revealed *P <0.05; †P < 0.01, ‡P < 0.001. Comparison of mice treated with othertreatments revealed polyclonal human IgM P = 0.05, sHIgm 46 P < 0.05.All other comparisons were not statistically significant. There was nodifference in the CNS-type remyelination between polyclonal human IgM,sHIgM22, and sHIgM 46. Area of peripheral nervous system-type SCremyelination ranged from 0 to 0.08 mm². This corresponded to 0.0 to6.92 percentarea of peripheral nervous system type SC remyelination as afunction of myelin pathology. There was no statistical difference in thearea of myelin pathology in the various treatment groups or compared toPBS or in other peripheral nervous system-type SC remyelination betweengroups.

Treatment with polyclonal human IgM resulted in more OL remyelinationthan that observed in mice treated with IVIg (p=0.05, FIG. 43A, B).Approximately one quarter of the total area of myelin pathology wasremyelinated in mice treated with polyclonal human IgM, representingthousands of ensheathed axons. On average, 1 mm2 within confluentlyremyelinated areas of pathology (FIG. 43B) corresponded to 46,000 to125,000 remyelinated axons. Therefore, the CNS remyelination followinghuman Ig treatment was extensive. Few inflammatory cells or macrophageswere present. In contrast, in mice treated with PBS, areas of myelinpathology contained few remyelinated axons (FIG. 43H). Signs of activemyelin destruction, such as myelin whirls, inflammatory cells andmacrophages were present.

As an additional, faster, method to judge the effectiveness of atreatment to promote remyelination the 10 spinal cord sectionsrepresentative of an animal were examined for the presence of areas ofwhite matter pathology that demonstrated nearly complete repair. Wedefined complete repair as an area of white matter pathology with nearlyconfluent remyelinated axons and no inflammatory cells or macrophagespresent (as in FIG. 43 B, F, G), a very rare event in spontaneousremyelination. At least one area of complete repair was observed in fourof ten animals treated with IVIg and in ten of fourteen animals treatedwith polyclonal human IgM. We concluded that both IVIg and polyclonalhuman IgM promote remyelination compared to PBS treatment and thatpolyclonal human IgM is superior to IVIg in the ability to promote CNSremyelination.

Human mAbs That Bind to OLs Promote CNS remyelination in TMEV-infectedMice

All of the previously identified mouse mAbs that promote CNSremyelination bind to OLs (23,26). To screen human mAbs for testing inthe TMEV model, human mAbs were tested for the ability to bind to thesurface of rat OLs in unfixed mixed primary glial culture. Primarycultures established from neonatal rat brain contain OLs at varyingstages of differentiation at 9 days in vitro (38). Our sources of humanmAbs were serum-derived human monoclonal IgMs (sHIgMs) and sera-derivedhuman monoclonal IgGs (sHIgGs). None of 50 sHIgGs bound to unfixed ratOLs, but six of 52 sHIgMs bound to the surface of rat OLs co-labeledwith the anti-sulfatide mAb, O4 (39).

The six OL-binding sHIgMs were used to treat TMEV-infected mice. Groupsof five animals each received a single injection of 0.5 mg of human mAb.The average percent area of OL remyelination following treatment withsHIgM22 and sHIgM46 (FIG. 43 F, G) were both significantly above thebackground levels attributable to spontaneous remyelination. The otherfour OL-binding sHIgMs promoted remyelination at levels comparable to orbelow the level observed following treatment with PBS. A second set ofanimals were treated with sHIgM22, sHIgM46 or PBS to confirm the initialobservations. SHIgM14 was also repeated as an example of a human mAbthat bound to OLs, but did not promote remyelination. The combined dataare presented in Table 1. Only animals that contained at least 5% totalwhite matter pathology were included in statistical analysis.

The highest percent area of OL remyelination was observed in animalstreated with sHIgM46 (27.1%), followed by animals treated with sHIgM22(17.1%). The percent area of remyelination following treatment withsHIgM14 (8.41%) was similar to that observed following treatment withPBS (6.74%). To test if any sHIgM, irrespective of antigen specificity,could promote remyelination we studied two mAbs in vivo whichdemonstrated no immunoreactivity to OLs in mixed primary culture, sHIgM1and sHIgM2 (FIG. 43C, D). The percent area of remyelination followingtreatment with sHIgM1 (8.3%), sHIgM2 (11.4%) were not significantlydifferent from the sHIgM14 or PBS treatment groups. In all groups theareas of white matter and areas of white matter pathology were notstatistically different. Compared to the remyelination observed in thePBS-treated group, the percent area of remyelination following treatmentwith sHIgM46 or sHIgM22 resulted in p values of <0.001 and <0.05,respectively. The area of peripheral nervous system-type SCremyelination ranged within treatment groups from 0 to 0.08 mm2. Thiscorresponded to values of 0.0 to 6.92 percent area of SC remyelinationas a function of white matter pathology. There were no statisticaldifferences in the percent area of SC remyelination between anytreatment group.

Comparing the percent area of OL remyelination observed followingtreatment with either human polyclonal or monoclonal preparationsrevealed that sHIgM46 was statistically superior to IVIg (p<0.05), butnot to polyclonal human IgM. The percent area of OL remyelinationobserved following treatment with sHIgM22 was no different than thatfollowing treatment with IVIg, polyclonal human IgM or sHIgM46.

When examined for areas of white matter pathology with complete repairat least one area was observed in four out of eight animals treated withsHIgM22 and in five out of five animals treated with sHIgM46. Incontrast, none of the animals treated with sHIgM1, sHIgM2, sHIgM14 orPBS contained a single area of complete repair.

Human mAbs, but not Polyclonal Human Igs, Bind to Rat Human OLs

If human mAbs are to be a potential therapy to promote remyelination inhumans, a reactivity to surface antigens on human OLs may proveimportant in targeting to areas of human CNS pathology. Therefore, wedetermined whether human remyelination-promoting mAbs could bind to OLsobtained from the adult human brain. Human glial cell cultures wereestablished from adult temporal lobe biopsies and immune-labeled withthe human mAbs at several time points in culture.

Three of the six sHIgMs that bound to the surface of OLs in our initialscreen, also bound to human OLs. At one week in culture morphologicallyimmature sulfatide positive human OLs labeled with sHIgM14 and sHIgM46,but not with sHIgM22. By 3 weeks in culture, morphologically complexsulfatide positive human OLs co-labeled with sHIgM14, sHIgM22 andsHIgM46 (FIG. 44A, B, C). By 4 weeks in culture, virtually allMBP-positive human OLs also bound sHIgM22 and sHIgM46, but the bindingof sHIgM14 was greatly reduced (data not shown).

Neither IVIg nor polyclonal human IgM bound to the surface of human OLsin culture at any time tested. However, polyclonal human IgM boundstrongly to white matter tracts and a variety of neuronal populationswhen incubated with fresh unfixed slices of rodent CNS. IVIg wascompletely negative in this binding assay (data not shown). SHIgM22 andsHIgM46, both of which promoted remyelination, and sHIgM14, which didnot promote remyelination, also bound to the surface of purified myelinbasic protein-positive rat OLs (data not shown).

We concluded that an affinity for OL antigens may be necessary, but isnot sufficient for a human mAb to promote remyelination. The fact thatboth human mAbs that promote significant remyelination bind to maturedifferentiated human OLs underscores the possible requirement of mAbs tobe directed against surviving adult OLs for in vivo function.

To exclude the possibility that human Igs or mAbs promoted remyelinationby neutralizing virus, each preparation was tested for reactivity topurified TMEV antigens by Western blotting (34). None of the human Abpreparations reacted with TMEV proteins; however, rabbit polyclonal Igraised against TMEV reacted strongly to four virus capsid proteins (datanot shown).

Peripheral B-cells were obtained from the individual from which sHIgM22was identified. The light and heavy chain variable domain sequences ofsHIgM22 were determined. The sHIgM22 light chain variable region(GenBank accession AF212992) belongs to the λ subgroup I of the humanlight chain variable regions. The sHIgM22 heavy chain variable region(GenBank accession AF212993) belongs to subgroup III of the human heavychain variable regions. There were significant differences between thesHIgM22 variable domains and the closest known human germline variabledomain sequence (40).

Discussion

In this series of experiments we have demonstrated that human Abs canpromote CNS remyelination. More extensive remyelination was observed inthe spinal cords of TMEV-infected mice following treatment withpolyclonal human IgM than treatment with human IVIg. In addition, weidentified two human monoclonal IgMs that consistently enhancedremyelination. Both mAbs were isolated from the sera of patients withWaldenstrom's macroglobulinemia (WM), a class of lymphoma characterizedby the malignant clonal expansion of a single B-cell at the late stageof maturation which floods the serum with a monoclonal IgM (41). Thehigh level of these mAbs do not appear to be deleterious. In patientswith WM the dominant IgM normally recognizes antigens that arerecognized by the IgM repertoire present in healthy individuals (42).Our ability to readily identify and isolate OL antigen-binding,remyelination promoting mAbs from the human population lends support tothe concept that these Abs are common among the B-cell repertoire andmay function as modifiers in response to CNS injury.

Remyelination-promoting mAbs May be Produced in the Sera of Individualswhen Confronted with CNS Damage.

Although both IVIg and polyclonal human IgM promoted remyelinationneither bound to rat or human OLs in culture. In contrast, both humanmAbs that promoted remyelination bound to both rat and human OL surfaceantigens. The increased efficacy of human mAbs to promote remyelinationmay be due to the effective targeting to adult OLs in the area ofdamage. Stangel reported that IVIg had no affect on the differentiation,migration or proliferation of OL progenitors in culture; however, thebinding of IVIg to OL progenitors was not assessed (43). The lack ofaffinity of IVIg to OLs likely explains the lack of any discernibleaffect on OL progenitors. Nevertheless, the fact that IVIg does not bindto OLs implies that the mechanism of action in promoting remyelinationmay be distinct from that of the human mAbs.

The very same preparation of polyclonal human IgM used in this study hasbeen demonstrated to neutralize autoantibodies (28) and alter cytokineexpression in EAE (44) and to be beneficial in a mouse model ofmyasthenia gravis (45). Polyclonal human IgM, but not IVIg, binds tomyelinated tracts in unfixed slices of rodent brain. Neither polyclonalpreparation bound to fresh human white matter. Polyclonal human IgM maypromote significant remyelination in the mouse via a combination ofgeneral immunoregulation, binding to pathogenic antibodies andopsonization of myelin debris.

The mechanism by which Igs promote remyelination remains to beelucidated. Since many of the remyelination-promoting mAbs bind to OLsand/or myelin, it is reasonable to hypothesize a direct effect on therecognized cells. There are examples of mAbs binding to and altering thebiology of OLs in culture (46-48). However, since the mAbs that promoteremyelination have varying specificities (23, 26) it is unlikely thateach mAb functions directly through a common antigen or receptor. Apolyvalent molecule like an IgM could bring normally disparate signalingmolecules into close proximity within the plasma membrane withsubsequent activation (49). Since most of the remyelination-promotingmAbs appear to bind to lipids (26), the binding of these IgMs to thecell surface could reorganize the plasma membrane and facilitate asignaling pathway. When SCH94.03 is added to mixed primary glialcultures a 2-3 fold increase the uptake of tritiated thymidine isobserved (Rodriguez, unpublished observations), but the exact identityof the proliferating cells remains to be determined.

Another potential mechanism by which remyelination-promoting mAbs mayfunction is by targeting to myelin debris or damaged OLs. Binding to OLsor myelin may enhance the clearance of cellular debris from areas ofdamage, allowing the normal process of spontaneous CNS repair toprogress. Perhaps the mechanism of action of polyclonal human Igs isprimarily through immunomodulation-via an inhibition of B-celldifferentiation or an alteration of cytokine expression and theanti-idiotypic network (27, 50)-whereas the action of the human mAbs isvia a direct targeting to OL antigens and/or myelin. No characteristicwas completely predictive of an Ab's ability to promote remyelination.In fact, one human mAb tested in chronically TMEV-infected mice appearsto suppress remyelination below the level of spontaneous remyelination,suggesting that certain OL-binding human mAbs can inhibit remyelinationin vivo or may exacerbate demyelination. This is consistent with theobservation that specific mAbs reactive to OL antigens (i.e., myelinoligodendrocyte glycoprotein, 51) enhance demyelination in EAE (52).Ultimately, proof of an Ab's remyelinating potential and lack ofpathogenicity requires in vivo testing.

Several double-blind, placebo-controlled trials with human IVIg haveshown some efficacy in MS (18,36,37). Polyclonal human IgM, sHIgM22 andsHIgM46 all enhanced CNS remyelination in the TMEV model as well asIVIg, suggesting that these Abs may be as effective in MS. Human mAbsthat bind to OLs may have the additional benefit of direct OLstimulation. Human mAbs can be produced free from potential pathogeninfection and can be structurally altered to augment their effectivenessand immunogenicity. In contrast to mouse mAbs or “humanized” mouse mAbs,human mAbs should result in minimal immune response and are readilyapplicable to human trials. Given that human mAbs promoted remyelinationin chronically paralyzed animals provides hope that successful therapiescan be developed for patients with long-standing disabilities.

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Example 8

As described in the prior Examples, we have used two approaches toidentify human monoclonal antibodies that induce a similar pattern ofremyelination in the Theiler's virus model of demyelinating disease. Thefirst approach was to transform human B cells with Epstein Barr Virus(EBV) to generate immunoglobulin secreting B cell clones. The resultingcell lines were screened to identify cultures that expressed high levelsof antibody and the ability of the produced antibodies to bind CNSantigens, with particular emphasis on those that bound oligodendrocytes.The second approach was to perform a similar screen CNS-binding of serumfrom patients diagnosed with a monoclonalgammopathy such as MUGUS,lymphoma, or Waldenstrom's syndrome. In the case of the EBV transformedcells, the cells themselves might provide a source of antibody forgenerating sufficient quantities of GMP grade antibodies for clinicaltrials. In addition, antibodies identified from either source could beproduced more optimally in an artificial antibody producing system usingsynthetic antibody genes encoding the antibodies of interest. Weanticipate that hybridoma cell lines transfected with an antibodyexpression cassette encoding the antibody of interest will providesufficient antibody of interest for in vivo analysis and clinicaltrials.

In the course of the screening studies, we have identified a set ofhuman monoclonal IgM antibodies that induce statistically significantremyelination in our in vivo Theiler's virus model of demyelinatingdisease (TABLE 11). Each of these antibodies mimicked the remyelinationresponse originally described with the prototypical murine monoclonalantibody SCH 94.03. Among these human antibodies are two derived fromEBV-transformed B cell lines, designated MSI 19-D10- and CB2b-G8, andtwo antibodies, designated sHIgM22 and sHIgM 46, identified among apanel of antibodies from more than 50 patients expressing high levels ofmonoclonal IgM in their sera.

TABLE 11 Remyelination induced by human monoclonal antibodies in SJL/Jmice chronically infected with Theiler's virus. Treatment %Remyelination Statistical Evaluation Comparison 1 PBS (n = 7)  6.74(+/−1.80) sHIgM22 (n = 8)  17.6 (+/−3.42) P < 0.05  sHIgM 46 (n = 5)27.12 (+/−4.01) P < 0.001 Comparison 2 PBS (n = 12)  8.25 (+/−1.44) MSI19-D10 (n + 13) 24.38 (+/−2.91) P < 0.001 CB2b-G8 (n = 12) 23.51(+/−3.13) P < 0.001 Animals were chronically infected with DA strain ofTheiler's virus for greater than 9 months prior to treatment with asingle ip injection of 0.5 mg IgM antibody isolated from patient serum(sHIgM22 and sHIgM 46) or EBV transformed cell lines (MSI 19-D10 orCB2b-G8). Five weeks later, the mice were perfused with fixative andspinal cords were isolated for histological analysis. Areas ofdemyelination and remyelination were assessed directly by microscopy.Percent area remyelinationwas determined by the formula arearemyelinated/area demyelinated × 100. Treatment effects were evaluatedby statistical comparison to groups of animals that received PBSinjections instead of antibody.

The structures of the IgM heavy and light chains for both the antibodiesderived from the EBV-transformants have been determined by analysis ofcDNA generated from immunoglobulin mRNA isolated from the cells. Thesequences of the heavy and light chain variable regions of MSI 19-D10and sHIgM22 are provided in FIGS. 35-38 (SEQ ID NOS: 11, 12, 13, 14 and7, 8, 9 and 10). The sequences of the heavy and light chain variableregions of CB2b-G8 are provided in FIGS. 45 and 46 (SEQ ID NOS: 15, 16,17, 18). The sequences themselves are not remarkable other than theydiffer somewhat from known germline immunoglobulin sequences. Thus, theymay be the products of somatic diversification during the course ofimmune responses against unidentified antigens. The value of thesequences is that they provide a blue print for the construction ofexpression vectors for the production of the immunoglobulin undercontrolled conditions.

Similarly, the structures of the heavy and light chains from the serumof one of the IgM-producing patients were determined by protein sequenceanalysis, followed by cloning and sequence analysis of cDNA fromperipheral blood mononuclear cells isolated from the patient. Twoclosely related heavy and light chains were identified in the patient'sserum, designated sHIgM22 (FIGS. 35 and 36) (SEQ ID NOS: 7, 8, 9, 10).The two heavy and two light chains were both present in the isolatedcDNA populations at a ration of 60:40. Both antibodies share a commonμ-VDJ rearrangement and λ-VJ rearrangement, indicting that they arederived from a common B cell precursor. They have subsequently diverged,as a result of the accumulation of mutations that have altered thestructures of their variable regions. We conclude that both antibodiesare expressed in the serum of the patient because peptides from bothantibodies were characterized from the protein isolated from the serum.However, the two distinct combinations of variable and light chains werenot observed directly, leaving open the possibility that othercombinations of the identified heavy and light chains may actually bepresent. Based on the positions of the observed amino acidsubstitutions, we suspect that the antibodies have very similarreactivity patterns.

Example 9 Development of a Transfection System for the Expression ofAntibody Genes in Cell Culture

In order to generate a renewable supply of high titer antibody from thehuman antibodies, we have developed a transfection-based expressionsystem for generation of recombinant antibody. Hybridoma cells whichhave been selected for the loss of endogenous immunoglobulin mRNAproduction can be transfected with recombinant antibody genes togenerate cells expressing the antibodies of interest.

We have explored the use of a series of genomic and cDNA based vectorsystems to express cloned antibody genes in cell culture. We havesuccessfully expressed light chain protein using either genomic orcDNA-based genes. We have achieved heavy chain expression using agenomic-based heavy chain vector PAC 4026 (kindly provided by Dr. SherieMorison at UCLA). However, the yield of antibody with this vector systemis too low for practical use in vivo. Our focus has been to develop anew vector that will routinely yield transfected hybridoma clones thatproduce high titer antibody. Our current strategy is to assemble thevector from components that individually have been shown to work well inour hands.

We have shown that a vector expressing dHfR and an immunoglobulin lightchain cDNA under CMV-promoter control expresses light chain and thatthis expression can be amplified by growing the transfectedimmunoglobulin producing cells in increasing concentrations ofmethotrexate (FIG. 47). We then clone this functional unit, expressingimmunoglobulin light chain and dHfR, into the vector expressing thegenomic heavy chain gene encoding the complementing μ chain. The firstvector we have created along this line encodes mouse/human chimeric94.03 and is shown in the top panel of FIG. 48. The vector has beenintroduced into antibody-negative hybridoma cells and clones expressingsmall amounts of functional antibody have been isolated. The antibodystains CNS tissue in a manner identical to the native mouse antibody94.03 (FIG. 49). These antibody-producing clones are now undergoingselection with increasing amounts of methotrexate to expand the amountof antibody being produced. Because we have identified two heavy and twolight chains in the human serum sHIgM22, all four permutations of heavyand light chain need to be evaluated. Vectors expressing three of thefour possible combinations of sHIgM22 identified in our sequence studyhave been prepared and are now being introduced into theimmunoglobulin-negative hybridoma cells. The prototype vector is shownin the bottom panel of FIG. 48. Recombinant sHIgM22 (22BII) comprisingClone B heavy chain and Clone II light chain (as denoted in FIGS. 35 and36) was generated based on the prototype vector. Recombinant 22BII isactive and demonstrates cell binding similar to native LYM 22.

Methods

Construction of Expression Vectors for Expressing Mouse/Human Chimeric94.03 (M1) and sHIgM 22 Antibody:

Assembly of Expression System for Mouse/Human Chimeric 94.03

The assembled vector consists of two units. The first encodes the heavychain of the immunoglobulin which is encoded by a genomic DNA-derivedimmunoglobulin gene. The vector is in part a derivative of a backbonevector (PAG 4026) obtained from the laboratory of Dr. Sherrie Morrisonfrom UCLA. The PAG 4026 vector encoded an IgM heavy chain expressing anirrelevant variable region. There were no convenient cloning sitesavailable for the substitution of variable regions of interest. Wetherefore engineered sites by deletion of the irrelevant heavy chainsequences and reconstitution of the regions flanking the variable regionwith unique restriction sites (Rsr II at the 5′ end and Pac I at the 3′end). As the sequence of the PAG vector was not known, we determinedwhich restriction enzyme sites would be unique by trial and error usingenzymes that recognize sequences infrequently present in mammalian DNA.

The heavy chain variable region of the mouse IgM monoclonal antibody94.03 was isolated from cDNA by PCR using the RsrII primerACTCCCAAGTCGGCTCGCTTTCTCTTCAGTGACAAACACAGACATAGAACATTCACCATGGGATGGAGCTGTATCACT (SEQ ID NO: 53) to introduce the RsrII siteupstream of the leader sequence and the PacI primerACTGACTCTCTTAATTAAGACTCACCTGAGGAGACTGTGAGAGTGGT (SEQ ID NO: 54) tointroduce the Pad site while maintaining the correct splice junction atthe 3′ end of the variable region coding block.

The second part of the expression vector is a derived from multipleplasmids. The finished construct contains EagI sites at both termini,the DHFR (dihydrofolate reductase) coding sequence under regulatorycontrol of the SV40 promoter and a chimeric mouse/human kappa lightchain cDNA coding block under regulatory control of the CMV promoter.This portion of the vector was assembled in a step wise fashion startingwith three plasmids (pClneo (Promega Corporation), pUC18 (New EnglandBiolabs), and pFR400 (Simonsen and Levinson, Proc Natl Acad Sci USA80:2495:1983)) that provided appropriate cloning sites, promoterregions, polyadenylation signals, and the DHFR coding block. After aseries of modifications which included the introduction of syntheticlinker regions and deletions of undesirable restriction endonucleaserecognition sites, the methotrexate selectable light chain cassette wasassembled. The cassette includes unique restriction endonuclease sites(Nhe I and Xho I) that flank the cDNA coding block of the light chaingene.

The chimeric light chain gene was assembled from two cDNA sequencesusing the PCR splicing by overlap extension technique (Horton et al.Gene 77:61:1989). The primers flanking the fused regions of the chimericcDNA (contained the enzyme recognition sequences for the endonucleaseXho I and Nhe I. The 5′ primer used to amplify the fused gene productwas TTGGCGCGCCAAAGACTCAGCCTGGACATGATGTCCTCTGCTCAGTTC (SEQ ID NO: 55);the 3′ primer was ATAGTTTAGCGGCCGCATTCTTATCTAACACTCTCCCCTGTTG (SEQ IDNO: 56). The cDNA coding block was inserted into the light chaincassette vector using these sites.

Once assembled, the cassette was excised using the endonuclease Eag Iand inserted into the unique Eag I site in the vector containing theheavy chain gene. The resulting construct contains the coding sequencesfor both the heavy and light chain components of the mouse/humanchimeric antibody for “humanized” 94.03. The heavy chain is expressed bythe human IgH promoter and the light chain is expressed by the CMVpromoter. The dHFR gene provides an amplification marker and isexpressed by the SV40 promoter. Each of these genes containspolyadenylation signals at the 3′ ends. Other important features of thevector include a bacterial origin of replication and a gene expressed inbacteria encoding resistance to ampicillin. The heavy variable and lightchain cDNA coding blocks are flanked by unique restriction endonucleasesites that can be use to substitute new immunoglobulin sequencesisolated from mRNA of any antibody producing cell or syntheticimmunoglobulin genes.

Insertion of sHIgM22 Sequences into the Expression Vector System

The cDNA of mRNA encoding the heavy and light chains of sHIgM22 wereprepared by PCR amplification of peripheral blood RNA using 5′ primersdeduced from amino acid sequence information and sequences in theconstant regions of the heavy and light chain respectively. The heavychain variable region coding block, leader sequence and donor splicejunction along with the flanking RsrII and Pac I sites were assembled byusing PCR to add the 5′ regionGACTCGGTCCGCCCAGCCACTGGAAGTCGCCGGTGTTTCCATTCGGTGATCATCACTGAACACAGAGGACTCACCATGGAGTTTGGGCTGAGCTGGGTTTTCCTCGTTGCTCTTTTAAGAGGTGTCCAGTGTCAGGTGCAGCTGGTGGAGTCTGG (SEQ ID NO: 57) and the 3′sequences CCTTAATTAAGACCTGGAGAGGCCATTCTTACCTGAGGAGACGGTGACCAGGGTT C (SEQID NO: 58). The resulting DNA molecule was digested with Rsr II and PacI and subsequently cloned into the expression vector, substituting thedesired variable region sequence for the irrelevant sequence in thevector.

The light chain sequence was assembled in two steps. The lambda constantregion was isolated from mRNA by RT-PCR using the 5′ primerCTAGCTAGCGTCCTAGGTCAGCCCAAGGCTGCCCCC (SEQ ID NO: 59) and 3′ primerATAGTTTAGCGGCCGCACCTATGAACATTCTGTAGG (SEQ ID NO: 60). This fragment wascloned using a unique AvrII site and a 3′ Not I site into the pCIneovector.

The variable region of sHIgM22 was generated by RT-PCR using the 5′primer CTAGCTAGCCCGAATTTCGGGACAATCTTCATCATGACCTGCTCCCCTCTCCTCCTCACCCTTCTCATTCACTGCACAGGGTCCTGGGCCCAGTCTGTGTTGACGCAGCCG (SEQ ID NO: 61)in order to introduce the needed Nhe I site and leader sequence onto thecDNA. The 3′ primer, GGGCAGCCTTGGGCTGAGCTAGGACGGTCAGC (SEQ ID NO: 62),was used to introduce an AvrII site so that this fragment could bejoined with the constant region piece. The resulting coding blockcontaining a functional leader signal was flanked by the necessary NheIand Xho I sites for cloning into the dHFR/light chain cassette, whichwas subsequently assembled with the heavy chain plasmid to generate thefinal product containing both the heavy and light chain coding sequencesand promoters needed for expression in mammalian cells.

Example 10 A 94.03 IgG Isotype Antibody

One strategy for determining the importance of isotype in the ability ofthe mouse antibody 94.03 to induce remyelination is to generate arecombinant antibody that expresses the variable region of 94.03 with anIgG isotype. As an alternative strategy, we sought to identify a naturalisotype switch variant within the population of 94.03-producing cells inculture. Spontaneous switch variants have been known to appear uponoccasion in cultures of this type. After successive FACS sorts of cellsstained for cell surface IgG, we were able to isolate a clonal cell linesecreting 94.03 bearing the IgG₁ isotype (FIG. 50). The structure of theantibody produced by these cells was confirmed by ELIZA, bycharacterization of the produced protein on SDS gels, and by cDNAcloning. Direct sequence analysis as outlined in FIG. 51 produceddefinitive data indicated that we have isolated an IgG₁ variant of the94.03 antibody.

Example 11 IgG₃ Isotype Anti-Oligodendrocyte Mouse Antibody 09

The mouse O9 antibody was isolated as an anti-oligodendrocyte antibodyand is of the IgG₃ subtype (Kuhlmann-Krieg, S., Sammer, I. and ShachnerM. (1988) Devel Brain Res 39:269-280). The O9 antibody binds stronglyand specifically to white matter in the CNS. We examined anddemonstrated the ability of the O9 antibody to stimulate remyelinationin the TMEV model. The O9 antibody heavy chain variable region sequenceis provided in FIG. 52 (SEQ ID NOS: 19 and 20). The sequence of thekappa light chain 1 variable region of 09 is provided in FIG. 53 (SEQ IDNOS: 21 and 22). The sequence of the kappa light chain 2 variable regionof 09 is provided in FIG. 54 (SEQ ID NOS: 23 and 24).

Example 12 IgM Monomers Induce Remyelination

Another approach to deciphering the importance of structural features ofthe IgM antibodies for the induction of remyelination is to fractionatethe antibody biochemically and to evaluate the ability of the antibodyfragments to induce remyelination in vivo. One possibility is that theidentified antibodies have low affinity for CNS structures, andtherefore, the decavalency of IgM may be critical for remyelinatingactivity because the multiple binding sites provide enough avidity forthe antibodies to interact with the target structures in the CNS. Toaddress this question, we have generated IgM monomers by reduction ofthe disulfied bonds that hold the pentameric immunoglobulin moleculestogether. The resulting monomers are divalent. The monomers fail to bindoligodendrocytes in vitro and do not stain brain sections in the patternobserved with the intact native antibody. However, the monomericantibodies retain the ability to induce remyelination in vivo (TABLE 12and TABLE 13). This is a notable result in light of the absence ofobserved staining in our in vitro assays. One possibility is that invitro assays monitoring binding are not as sensitive as the bioassay forremyelination. Because there is such a strong correlation betweenbinding and the induction of remyelination, we believe that thespecificity of these antibodies for CNS structures is important despiteour inability to observe binding with the monomers.

TABLE 12 Remyelination induced by monomeric fragments of murine IgM mAb94.03. Treatment % Remyelination Statistical Evaluation PBS (n = 7) 6.74(+/−1.80) Monomeric 94.03 (n = 8) 17.32 (+/−2.67)  P < 0.01 Pentameric94.03 (N = 5) 18.1 (+/−5.76) P < 0.01 IgM antibodies were reduced inmild conditions and alkylated. This treatment disrupted the pentamericstructure of the antibodies and allowed divalent monomers to be isolatedby column chromatography. Chronically infected SJL/J mice received atotal of 0.5 mg of antibody administered ip twice a week over the fiveweek treatment period. After five weeks, the animals were perfused withfixative and their spinal cords removed for histological analysis.Percent remyelination was determinedmicroscopically by comparing thearea of remyelinated lesions to total demyelinated area as indicated inTable 1. Individual treatment groups were compared to animals whichreceived PBS injections instead of antibody.

TABLE 13 CNS Remyelination by Mouse Monoclonal Antibodies Area ofCNS-Type Comparison of % Area of White Area of Myelin RemyelinationRemyelination to Treatment No. of Mice Matter (mm2) Pathology (%) (%)PBS Group Pentameric 94.03 5 9.7 ± 1.2 14.2 ± 3.6  18.1 ± 5.8 P = 0.05 Monomeric 94.03 8 9.3 ± 1.0 9.3 ± 1.2 17.3 ± 2.7 P = 0.007 Expt 1 & 2Monomeric 94.03 5 9.8 ± 1.3 9.1 ± 1.1 19.1 ± 3.4 P = 0.006 Expt 1Monomeric 94.03 3 8.4 ± 1.5 9.8 ± 2.9 14.4 ± 4.5 P = 0.087 Expt 2 PBS 79.8 ± 0.6 11.9 ± 1.8   6.7 ± 1.8 Values represent the mean ± standarderror of the mean. Statistics by t test of the percentage of area ofCNS-type remyelination per area of white matter pathology in micetreated with monoclonal antibodies as compared to those treated withPBS. Only animals with area of white matter pathology ≧5% were includedin statistical analysis.

We have further fractionated the antibody by generating (Fab′)₂ Fab, andFv fragments of the antibody. SJL mice chronically infected withTheiler's virus have been treated with these antibody fragments todetermine whether divalent fragments missing the Fc portion of theantibody or monovalent antibody fragments comprised primarily of asingle antigen binding site can induce remyelination.

A parallel analysis of the human monoclonal antibody sHIgM22 wasperformed to determine whether antibody fragments of this human IgMbehave in a similar manner. Preliminary analysis indicates that the(Fab′)₂ fragments of sHIgM22 induce remyelination. If it can bedetermined that a single small binding domain can induce remyelination,this information may prove important for determining the mechanism ofrepair as well as provide an avenue for the development of anpharmacological analogue.

Example 13 sHIgM 46 Antibody Induces Myelin Repair

Our initial studies suggests that the inductin of myelin repair by sHIgM46 may be qualitatively superior to the repair observed with sHIgM22.Upon histological examination of sections of SJL mice that werechronically infected with TMEV, smaller areas of demyelination wereobserved following treatment with sHIgM 46 than with other monoclonalantibodies (TABLE 14). This observation was highly statisticallysignificant. This result is notable because treatment with the antibodydoes not begin until demyelinated lesions are well established and havereached maximum size in chronically infected animals. Our interpretationof this result is that myelin repair is so complete in some areas of thespinal cord that they are not being distinguished from normal areas ofthe cord during our standard histological examination. The lesions insHIgM 46 treated mice are examined by electronmicroscopy to confirmwhether the repaired lesions contain higher numbers of myelin wraps thanin other treatment groups.

TABLE 14 Qualitative differences in myelin repair by human antibodysHIgM 46 % White Matter Treatment Demyelinated Statistical EvaluationsHIgM 46 (n = 15)  4.07 (+/−2.52) All other antibodies (n = 70) 10.41(+/−6.26) P < 0.001 SJL/J mice chronically infected with Theiler's virusfor more than 9 months were divided into groups and individual groupsreceived a single 0.5 mg ip injection of one of a battery of monoclonalantibodies. After five weeks animals were perfused with fixative andtheir spinal cords were isolated for histological analysis. The area ofdemyelination was determined by measuring the total area of the cordoccupied by white matter and the area of demyelination visualized bylight microscopeusing 25X optics. The data are comprised of mice fromthree independent experiments. Antibodies used to treat animals in thepooled treatment group (“all other antibodies”) were human monoclonalIgM sHIgM 12, 14, 22, 47, 50, AKJR8, MSI 10E10, 2B2GE7, NA8FE4, andmouse antibodies O6, O9, RIP, and MOG. The data set passed normalitytests and were analyzed by ANOVA.

Example 14

The sequences of the heavy and light chain variable regions of humanantibodies AKJR4, CB2iE12 and CB21E7, and the light chain variableregion of MSI19E5 were determined. The sequences of the heavy and lightchain variable region of AKJR4 are shown in FIGS. 55 and 56,respectively (SEQ ID NOS: 25, 26 and 27, 28). The sequences of the heavyand light chain variable region of CB2iE12 are shown in FIGS. 57 and 58,respectively (SEQ ID NOS: 29, 30 and 31, 32). The sequences of the heavyand light chain variable region of CB21E7 are shown in FIGS. 59 and 60,respectively (SEQ ID NOS: 33, 34 and 35, 36). The sequence of the lightchain variable region of MSI19E5 is shown in FIG. 61, respectively (SEQID NOS: 37 and 38).

Example 15

A series of mouse and human antibodies were tested for their ability toinduce or generate calcium flux in mixed primary glial cultures(composed of astrocytes and oligodendrocytes) using the method providedabove in Example 5. The results (as a percentage of cells showingcalcium flux) for these antibodies are tabulated in TABLE 15, along withremyelination and oligodendrocyte surface immunoreactivity for eachantibody.

Antibodies demonstrating intracellular calcium changes are also noted aspromoting remyelination.

TABLE 15 CNS Reactive Antibodies Mediate Intracellular Calcium Changes #of responding Oligodendrocyte Treatment cells (mixed RemyelinationSurface (10 □g/ml) cell cultures) Promotion Immunoreactivity SCH 94.0336/251 (14%) * + + (3 □g/ml) SCH 79.08 25/137 (18%) * + − sHIgM22 33/272(12%) * + + (3 □g/ml) sHIgM50 12/222 (5%) * ? + 04 49/265 (18%) * + +CB2BG8 43/269 (16%) * + + AKJR8 49/244 (20%) * +/− (p = .06) − 94.03monomer  3/310 (1%) * + − CH12  0/203 (0%) − − sHIgM12  0/247 (0%) − −sHIgM14  0/203 (0%) − + sHIgM47  0/177 (0%) − + AKJR4  1/268 (0.4%) −

Example 16

Remyelination studies in TMEV-infected mice were performed with mouseantibody O9 versus monomeric and pentameric 94.03. The results of thesestudies are shown in TABLE 16.

Similarly, remyelination studies were performed with human antibodiesAKJR4, AKJR8 and MS110E10 and are shown in TABLE 17. Remyelination withMSI10E10 is significant.

TABLE 16 CNS Remyelination by Mouse Monoclonal Antibodies Area ofCNS-Type Area of CNS-Type Area of White Area of Myelin RemyelinationRemyelination Treatment No. of Mice Matter (mm2) Pathology (mm2) (mm2)(%) Pentameric 94.03 5 9.68 ± 1.27 1.47 ± 0.42 0.26 ± 0.11 18.1 ± 5.76Monomeric 94.03 8 9.30 ± 0.96 0.85 ± 0.12 0.13 ± 0.02 17.32 ± 2.67  09 58.44 ± 0.96 1.35 ± 0.33 0.36 ± 0.13 27.4 ± 6.33 PBS 7 9.78 ± 0.60 1.20 ±0.22 0.06 ± 0.02 6.74 ± 1.80 Values represent the mean ± standard errorof the mean. Statistics by t test of the percentage of area of CNS-typeremyelination per area of white matter pathology in mice treated withmonoclonal antibodies as compared to those treated with PBS revealed,monomeric 94.03 p = 0.007, 09 p = 0.005. Only animals with areas ofwhite matter pathology ≧5% were included in statistical analysis.

TABLE 17 CNS Remyelination in TMEV-Infected Mice Following Treatmentwith Human Monoclonal Antibodies (Oct 2000) Area of CNS-Type Area ofCNS-Type p value Area of White Area of Myelin RemyelinationRemyelination compared Treatment No. of Mice Matter (mm2) Pathology(mm2) (mm2) (%) to PBS AKJR4 4 8.78 ± 0.8 1.1 ± 0.2 0.05 ± 0.03  4.2 ±2.0 0.160 AKJR8 6 10.0 ± 0.7 1.0 ± 0.1 0.12 ± 0.02 13.3 ± 2.2 0.066MSI10E10 5  9.3 ± 1.4 1.9 ± 0.6 0.18 ± 0.04 11.3 ± 3.7 0.360 PBS 12  9.9± 0.4 1.1 ± 0.1 0.07 ± 0.01  8.3 ± 1.4 Values represent the mean ±standard error of the mean. Statistics by t test of the percentage ofarea of CNS-type remyelination per area of white matter pathology inmice treated with human bodies as compared to those treated with PBS.Only animals with areas of white matter pathology ≧5% were included instatistical analysis.

Example 17 Antibodies Promote Glial Cell Proliferation

The ability of mouse and human antibodies to promote glial cellproliferation was assessed. Mixed primary cultures of rat glia weregrown to 8 days in culture in a minimal media of DMEM, insulin and aminoacids. Antibodies were added to the media at the indicatedconcentrations for 24 hrs. Cultures were then pulsed with tritiatedthymidine for 24 hrs. Cell monolayers were dislodged with trypsin andcounted. PDGF and FGF are the positive control. The upper panel of FIG.63 shows that 94.03 promoted glial cell proliferation over media aloneand equal to that observed with the PDGF/FGF combination. In the lowerpanel of FIG. 63 it is shown that sHIgM22 and RsHIgM22 both promoteglial proliferation over media alone.

FIG. 64 depicts the analysis of promotion of glial cell proliferation byhuman antibodies native LYM 22, 22BII (recombinant sHIgM22 composed ofheavy chain Clone B and light chain Clone II), AKJR4 and AKJR8. Bothnative LYM 22 and recombinant 22BII promote glial cell proliferation.

Mouse antibody O9 induces glial cell proliferation at 1 μg/ml and 0.10μg/ml, as shown in FIG. 65.

Example 18 Recombinant sHIgM22 (22BII) is Specific for White Matter

To assess the cell binding characteristics of recombinant LYM 22(22BII), as compared to native LYM 22, slices of fixed postnatal ratcerebellum were incubated with serum derived human antibody 22 (sHIgM22)or the recombinant version (RsHIgM22) (22BII). As previously described,sHIgM22 binds to white matter tracts and to cells within the externalgranular and molecular layers. In contrast, RsHIgM22 is very specificfor only white matter. It is likely that polyclonal human IgMs presentin sHIgM22 account for the weak binding to other epitopes. The resultsare shown in FIG. 66.

Example 19 Mouse IgM Antibody Sequences

Upon examination of the cells for other purposes, we noted that the DNAsequences originally identified for certain mouse remyelinatingantibodies, and published, were incorrect (Asakura et al. (1995) Mol.Brain. Res. 34:283-293). Specifically, kappa chain sequences originallyidentified for the O1, O4, O9, A2B5 and HNK-1 antibodies were notcorrect. Each of the antibody producing cells were reanalyzed and thenew antibody sequences were cross-checked using sequence specificoligonucleotide probes in a Northern Blot study to confirm the identifyof the cells making the antibodies of interest. These correct sequencesare appropriate and should be utilized in humanized recombinantantibodies.

O9 Kappa Chain Sequence:

O9 hybridoma produces two light chains. One of them (Noted Above and inFIG. 53 (SEQ ID NOS: 21 and 22) as “O9 kappa light chain 1”) isubiquitous for all O-series hybridomas and originates from MOPC21 fusionpartner. This light chain does not appear to be important for theantibody activity of interest. The sequence of the O9-characteristickappa chain (noted as “O9 kappa light chain 2” and provided in FIG. 54(SEQ ID NOS: 23 and 24)) remains unchanged and is the correct 09 kappachain sequence.

O4 Kappa Chain Sequence:

The correct and complete O4 kappa chain sequence is shown in FIG. 67(SEQ ID NOS: 41 and 42).

O1 Kappa Chain Sequence:

This sequence, provided in FIG. 68 (SEQ ID NOS: 43 and 44) is completelynew. The previously reported O1 kappa chain was the shared MOPC21 kappachain which is also produced by the O1 hybridoma.

HNK-1 Kappa Chain Sequence:

The published HNK-1 sequence and the newly obtained sequence differ intwo nucleotides: 174 (G-C) and 281 (C-T, this changes the amino acidfrom S to F). The changes are highlighted on the sequence provided inFIG. 69 (SEQ ID NOS: 45 and 46).

A2B5 Kappa Chain Sequence:

This sequence of A2B5 kappa chain shown in FIG. 70 (SEQ ID NOS: 47 and48) is completely new. The previously reported A2B5 kappa chain sequenceis in fact the O4 kappa chain sequence.

Example 20 Sequence of sHIgM46 (LYM46) Antibody and RecombinantExpression of LYM22 and LYM46

The sequence of LYM46 was determined. The amino acid sequence (SEQ IDNO: 49) and nucleic acid sequence (SEQ ID NO: 50) of the LYM46 heavychain are depicted in FIG. 71. The amino acid sequence (SEQ ID NO: 51)and nucleic acid sequence (SEQ ID NO: 52) of the LYM46 heavy chain aredepicted in FIG. 72.

Various vectors have been generated and are being utilized in generatingrecombinant LYM46 and recombinant LYM22 and are depicted in FIG. 48B andFIGS. 73 through 79. Recombinant LYM 46 was generated using vectorpUD46M (depicted in FIG. 75). Recombinant LYM46 protein was tested usingisotype specific antibodies to confirm its kappa type. Recombinant LYM46stains equivalently to the isolated sHIgM46 from the patient onimmunostaining.

The vectors and recombinant antibody expression constructs weregenerated as follows:

Construction of pUD22BIIM (FIG. 74)

The plasmid puD22BIIM is related to pADM22 (also called pAGDF22—FIG.48B). All the functional elements (IgM heavy chain, lambda light chain,and dHfR gene cassette) are identical. The backbone plasmid of pAD wasexchanged for the traditional pUC18 plasmid backbone. This manipulationwas intended to simply and shorten the plasmid backbone of the vector.This vector has been used successfully to express small quantities ofLYM 22 (approximately 0.5 ug antibody/ml supernatant).

Construction of pUD46M (FIG. 75)

The plasmid pUD46M is closely related to the plasmid pUD22BIIM. Thevariable region of the LYM22 heavy chain gene was removed as a cassetteand replaced with the variable region sequence of the LYM46 sequence.The LYM22 lambda light chain sequence was replaced with the LYM46 kappalight chain sequence.

The Lym 46 heavy chain variable region sequence was synthesized usingoverlapping oligonucleotides and cloned into pUDM. The oligonucleotidesused were:

(SEQ. ID NO: 73) 5′ act ccc aag tcg gtc cgc ttt Template A--(SEQ. ID NO: 74) act ccc aag tcg gtc cgc ttt ctc ttc agt gac aaacac aga cat aga aca ttc acc ATG GAG TTT GGG CTGACC TGG CTT TCT CTT GTT GCT ATT TTA GAA GGT GTCCAG TGT GAG GTG CAG CTG GTG GAG TCT GGG GGA GGCTTG GTC CAG CCT GGG GGG TCC CTG AGA CTC TCC TGTGCA GCC TCT GGA TTC ACC TTT AGT AGC TAT TGG ATGACC TGG GTC CGC CAG GCT CCA GGG AAG GGG CTG GAG TGG GTG GCC AAC ATA AAGTemplate B-- (SEQ. ID NO: 75)CTG GAG TGG GTG GCC AAC ATA AAG AAA GAT GGA AGTGAG AAA TCC TAT GTG GAC TCT GTG AAG GGC CGA TTCACC ACC TCC AGA GAC AAC GCC AAG AAC TCA CTG TATCTG CAA ATG AAC AGC CTG AGA GCC GAG GAC ACG GCTGTG TAT TAC TGT GCG AGA CCC AAT TGT GGT GGT GACTGC TAT TTA CCA TGG TAC TTC GAT CTC TGG GGC CGTGGC ACC CTG GTC ACT GTC TCC TCA ggt gag tct taa tta aga gag tca gt 3′primer-- (SEQ. ID NO: 76) act gac tct ctt aat tag 

The kappa light chain variable region was isolated as cDNA from patientperipheral blood mRNA using primers derived from the amino acidsequences from the predicted leader sequence of the light chain protein.The cDNA variable region sequence was fused to the kappa chain constantregion cDNA sequence using a unique internal restriction endonucleasesite Bsu36I. The oligonucleotide primers used to generate the variablesequence cDNA were as follows:

5′ primer containing the leader sequence (SEQ. ID NO: 77)CTA GCT AGC TCA AGA CTC AGC CTG GAC ATG GTG TTGCAG ACC CAG GTC TTC ATT TCT CTG TTG CTC TGG ATCTCT GGT GCC TAC GGG GAC ATC GTG ATG ACC CAG 3′ primer (SEQ. ID NO: 78)GAA CGC CTG AGG AGT ATT AT

The variable and constant region sequences were joined by ligation attheir common Bsu36I endonuclease cleavage site.

The assembled LYM 46 kappa light chain gene contained XhoI and NheIsites introduced in the flanking 5′ and 3′ ends during assembly. Theentire gene was cut out using these enzymes and then was ligated intothe pUDM vector to make the assembled pUDM46 vector system.

Assembly of pAD46M (FIG. 73)

The heavy and light chain sequences were subsequently used to assemblepADM46 in a fashion identical to that described for LYM 22.

Assembly of pUD22G1 and pUD22G2 Vectors (FIGS. 76 and 77)

cDNA sequences encoding the heavy chain regions of the IgG1 and IgG2isotypes were isolated from normal peripheral blood mRNA using theoligonucleotide primers:

5′ Primer for all human IgG subclasses with Bam HI site:(SEQ. ID NO: 79) CTG ATG CTA CGA TGG ATC CGC CTC CAC CAA GGG CCC ATC 3′Primer for gamma 1, and 2 with Sal 1 site: (SEQ. ID NO: 80)GCA TGA GTC TGA CAG CTG TTT ACC CGG AGA CAG GGA GAG GCT

These sequences were modified at their 5′ and 3′ ends by adding AscI andBsiWI restriction endonuclease sites. They were cloned into the pUDvector series by substituting the genomic IgM constant region exons withthe cDNA sequences encoding the IgG1 or IgG2 constant region cDNAsequences. A polyadenylation site was introduced 3′ to the codingsequences. The resulting vectors were designated pUD22G1 and pUD22G2respectively

These vectors produced small quantities of LYM 22 antibodies of the IgG1 or IgG 2 isotypes (<1 ug/ml of culture supernatant.)

Example 21 Expression of an IgG G1 and G2 Form of LYM22 and LYM46

Vectors to generate recombinant LYM22 and LYM46 IgG subtype G1 and G2have been generated and are depicted in FIGS. 76-79. LYM22 sequenceswere inserted into vectors pAD22G1 and pAD22G2 (FIG. 76) at theRsrII/PacI and Xho/NheI sites, similarly as described above (Example 9).Recombinant G1 subtype antibody was recovered from pAD22G1 andrecombinant G2 subtype antibody was recovered from pAD22G2.

Example 22 LYM46 Promotes Demyelination Early after TMEV Infection

As previously described above, including in Example 13, smaller areas ofdemyelination were observed with LYM46 versus with other antibodies. Tofurther assess and evaluate this, SJL mice were infected with TMEV aspreviously described and were examined early (21 days) after infectioninstead of evaluating mice longer after infection (6 months), or onchronic infection. FIG. 80 depicts evaluation of percent demyelinationand percent inflammation 21 days post infection on treatment of micewith PBS, sHIgM22 and sHIgM46. The sHIgM46 treated group is differentfrom the sHIgM22 or PBS treated group and shows significantly lessdemyelination and less inflammation. These results indicate that sHIgM46is blocking demyelination from occurring in the TMEV model.

Example 23 Remyelination by LYM22 Fragments and Monomers

Lym22 Fv, F(ab) and F(ab)′₂ fragments and monomers were generated byenzymatic digestion or reduction and assessed for remyelination capacityin a TMEV model.

Fv, F(ab) and F(ab)′₂ fragments and monomers were generated using thefollowing materials and methods:

Fv Fragments Production

For Fv production isolated IgM in 20 mM acetate buffer, 150 mM NaCl, pH4.0 was digested with pepsin (Worthington, Lakewood, N.J.) in aIgM:pepsin ratio of 20:1 at 4° C. After 24 h the same (as initial)amount of pepsin was added and proteolysis was continued for one moreday and then the solution was clarified by centrifugation (at 4° C.).Pepsin digestion was stopped by bringing pH up to 8.0 with 2 M Tris, pH8.0, buffer and the solution was concentrated in a centrifugal filterdevice (Millipore, 5 kD cutoff) to the volume suitable for applicationon gel-filtration column (approx. 2 mL for bigger size column) and againclarified by centrifugation at 14000 rpm. The Fv fragments were isolatedfrom the mixture by chromatography on a Superdex-75 column (Pharmacia,Upsalla, Sweden) equilibrated with PBS. Analysis of the Fv fragments wasperformed at 15% SDS-PAGE. Generally 2 and sometimes up to 4 closelyspaced bands can be seen in the range from 10 to 15 kD. For Fv fragmentsthe upper band observed was VH while the lower one was VL.

F(ab′)₂ and Fab Production

For F(ab′)₂ and Fab production, isolated IgM in 100 mM acetate buffer,150 mM NaCl, pH 4.5 was digested with pepsin (Worthington, Lakewood,N.J.) in a IgM:pepsin ratio of 40:1 at 4° C. After 3 h, digestion wasstopped by increasing pH to 8.0 with 2 M Tris buffer, pH 8.0, clarifiedby centrifugation and the solution was concentrated in a centrifugalfilter device (Millipore, 10 kD cutoff) to the volume suitable forapplication on gel-filtration column (approx. 2 mL for bigger sizecolumn) and again clarified by centrifugation at 14000 rpm. F(ab′)₂ andFab fragments were isolated by size-exclusion chromatography on aSuperdex-75 column (Pharmacia, Upsalla, Sweden) equilibrated with PBS.

Alternatively digestion with trypsin can be used as follows:

IgM in 75 mM Tris, 150 mM NaCl, 12 mM CaCl₂, pH 8.2 and IgM:trypsin(Worthington, Lakewood, N.J.) at ratio 20:1 was digested 4 h at 55° C.Before adding CaCl₂ IgM solution should be free of phosphates (i.e. fromPBS) since Ca-phosphate which will form and precipitate thus changingthe composition of the buffer. After cooling the proteolitic mixture onroom temperature, trypsin-inhibitor (from soybean; Worthington,Lakewood, N.J.) and phosphate buffer, pH 7.0, to 50 mM finalconcentration were added. Phosphate buffer was added only to precipitateCa⁺² and prevent its precipitation later in the column during thegel-filtration. The solution was cleared by centrifugation, concentratedand chromatographed over a Superdex-75 column. Identity of isolatedfragments was confirmed by SDS-PAGE in reducing and nonreducingconditions and ELISA with anti-human lambda chain MAb.

In the case that yield of Fab is low and that F(ab′)₂ is primaryproduct, an additional amount of Fab′ (which is basically the same thingas Fab) can be generated by reducing and alkylating F(ab′)₂ according tothe protocol for production of monomeric IgM written below.

Production of Monomeric IgM

Monomeric IgM was produced in 200 mM Tris, 150 mM NaCl, 1 mM EDTA, pH8.0, by reduction with 5 mM dithiothreitol (DTT) (Sigma), 2 h at roomtemperature in the dark and with occasional swirling. Subsequentalkylation was performed 1 h on ice by adding iodacetamide (IAA) to afinal concentration of 12 mM. Both DTT and IAA stock solution should befreshly prepared. IgM-monomers were isolated by size-exclusionchromatography on a Superdex-200 column equilibrated with PBS, andcharacterized by reducing and nonreducing 12% SDS-PAGE.

The results of assessment of remyelination by Lym22 versus monomers andfragments in a TMEV model are shown below in TABLE 18.

TABLE 18 Remyelination promoted by Lym22 fragments^(#) Quadrants withQuadrants with lesions remyelination Treatment No. of quadrants (%) (%)PBS n = 10 371 42.6 31.0 Lym22 n = 12 412 39.3 48.8 p = 0.002* Monomer n= 12 418 46.2 45.1 p = 0.01 F(ab′)2 n = 12 404 41.6 57.1 p < 0.001 Fab n= 12 397 41.3 37.2 p > 0.05 Fv n = 5 184 28.3 34.6 p > 0.05 Recombinant197 37.0 59.7 p < 0.001 Lym22 n = 6 ^(#)Results presented in the tableare summarized from two independent experiments performed fromindependent preps of Lym22 and its fragments. *Statistics was done bychi-square test.

Example 24 Antibody Induced Ca⁴⁵ Internalization

Various autoantibodies were tested for their ability to induce calciuminflux oligodendrocytes using labeled calcium Ca⁴⁵. While the earlierabove described calcium flux assay is a dynamic representation on a cellby cell basis of the presence or absence of calcium flux, this Ca⁴⁵internalization assay sums up the calcium influx over the assessmenttime and provides an influx signal. This influx can be used to measurethe amount of calcium flowing in based on the number of cells and theamount of calcium flux. The results of assessment of antibodies LYM22,LYM2, 94.03IgM, 94.03IgG, and LYM46 are depicted in FIGS. 83 and 84. Toassess calcium internalization, 10 ug/mL of each of the indicatedantibodies was applied to adherent CG4 cells for 15 min at 37 C in thepresence of Ca⁴⁵. Following calcium influx for this period of time,cells were chilled and washed extensively with LaCl₃ to chelate andremove any free calcium. Following washing, cells were lysed in 0.2 NNaOH and the lysates were assayed for cpm. The graph represents percentincrease in measured Ca⁴⁵ above background influx occurring in untreatedcells (i.e. cells only exposed to Ca⁴⁵ for 15 min in the absence ofantibody). The ionophore represents a maximal calcium influx responseinduced pharmacologically (i.e. all cells will flux calcium in responseto the ionophore). Error bars are standard error of the mean of fourseparate measurements.

As is evident from FIGS. 83 and 84, 94.03 IgM, LYM22 and LYM46demonstrate Ca⁴⁵ influx that is significant versus untreated cells.

Example 25 Antibody-Induced Protection from Apoptosis

Antibody-induced protection from H₂O₂-mediated apoptosis or cell deathwas examined. 10 ug/mL of antibody was applied to adherent CG4 cells inthe presence of varying concentrations of hydrogen peroxide (whichinduces a well-characterized apoptotic cell death) for 1 hr at 37° C.Following this incubation, cells were washed, fresh media was added, andan MTT assay was performed to measure cell viability and cell number.FIG. 85 indicates the relative number of cells surviving theH₂O₂-mediated insult in the presence of LYM22 versus the absence ofLYM22. At all concentrations of H₂O₂, LYM22 induced a small butsignificant increase in cell survival (FIG. 85). Similarly, antibody94.03 protected cells from H₂O₂ induced apoptosis (FIG. 86). Thepercentage of cells protected from apoptosis-induced cell deathcorrelates with the percentage of cells which show antibody surfacebinding by immunostaining in each case of LYM22 and 94.03 at theantibody concentration used in the MTT assay.

The MTT assay was performed as follows:

-   1. Prepare 10× MTT stock as 250 mg MTT in 50 mL PBS. Sterile filter    and store at 4° C.-   2. Prepare lysis buffer (SDS-DMF):-   100 g SDS-   250 mL N,N-dimethylformamide-   250 mL ddH2O-   3. Photograph wells prior to manipulation.-   4. Replace media in wells with 2 mL binding buffer (phenol    red-free).-   5. Add 200 uL stock MTT and incubate for 1 hr at 37° C.-   6. Remove media from wells by aspiration (take care not to dislodge    loosely adherent cells).-   7. If possible, wash well 1× with ice-cold PBS.-   8. Lyse cells in 1 mL SDS-DMF and incubate for 15 min at RT shaking    vigorously.-   9. Triturate lysate and transfer to microfuge tube. Centrifuge 2 min    at 16000 g to clear S/N.-   10. Read absorbance at 570 nm.    Reagents:-   MTT: Sigma #M2128, 250 mg-   3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide-   DMF: Sigma #D4254, 250 mL-   N,N-dimethylformamide

The above described MTT assay measures viable cells afterperoxide-induced apoptosis. The number of apoptosing cells can also bemeasured using known protocols or methods including the TUNEL assay(available from Roche Molecular Biochemicals Inc., catalog number1684809) or other reported and known apoptosis assays (Gavrieli Y. et alJ. Cell Biol. 1992 November; 119(3):493-501; Gorczyca W. et al CancerRes. 1993 Apr. 15; 53(8):1945-51; Gold R. et al Lab Invest. 1994 August;71(2):219-25).

The disclosure of the listed references provided in this section ofExamples as well as other publications, patent disclosures or documentsrecited herein, are all incorporated herein by reference in theirentireties.

This invention may be embodied in other forms or carried out in otherways without departing from the spirit or essential characteristicsthereof. The present disclosure is therefore to be considered as in allrespects illustrative and not restrictive, the scope of the inventionbeing indicated by the appended Claims, and all changes which comewithin the meaning and range of equivalency are intended to be embracedtherein. Those skilled in the art will recognize, or be able toascertain, using no more than routine experimentation, many equivalentsto the specific embodiments of the invention described herein. Suchequivalents are intended to be encompassed by the following claims.

What is claimed is:
 1. An isolated nucleic acid which encodes anantibody sHIgM22, a recombinant antibody derived therefrom, or a monomeror an active fragment of sHIgM22 capable of inducing remyelinationwherein the antibody, the recombinant antibody, monomer or activefragment comprises the heavy chain variable region CDR1, CDR2 and CDR3sequences as set out in FIG. 35 and SEQ ID NO:7 and the light chainvariable region CDR1, CDR2 and CDR3 sequences as set out in FIG. 36 andSEQ ID NO:9.
 2. The nucleic acid of claim 1 which encodes an antibody orrecombinant antibody comprising a heavy chain sequence comprising theheavy chain variable region sequence of SEQ ID NO: 7 and a light chainsequence comprising the light chain variable region sequence of SEQ IDNO:
 9. 3. The nucleic acid of claim 1 which encodes an antibody orrecombinant antibody comprising a heavy chain sequence comprising theheavy chain variable region sequence of the antibody produced by ATCCAccession No. PTA-8671 and a light chain sequence comprising the lightchain variable region sequence of the antibody produced by ATCCAccession No. PTA-8671.
 4. The nucleic acid of claim 1 which encodes anantibody produced by ATCC Accession No. PTA-8671, a recombinant antibodyderived therefrom, or a monomer or an active fragment of the antibodyproduced by ATCC Accession No. PTA-8671, capable of inducingremyelination, wherein the antibody, the recombinant antibody, themonomer or the active fragment comprises the heavy chain variable regionCDR1, CDR2 and CDR3 sequences and the light chain variable region CDR1,CDR2 and CDR3 sequences of the antibody produced by ATCC Accession No.PTA-8671.
 5. An isolated nucleic acid which encodes an antibody sHIgM22or a recombinant antibody derived therefrom having a heavy chainsequence comprising the heavy chain variable region sequence of SEQ IDNO: 7 and a light chain sequence comprising the light chain variableregion sequence of SEQ ID NO:
 9. 6. An isolated nucleic acid whichencodes the monoclonal antibody produced by ATCC Accession No. PTA-8671.7. The nucleic acid of claim 1, 5 or 6 which is a DNA sequence.
 8. Anisolated DNA sequence which encodes an antibody having a heavy chain anda light chain, said DNA sequence selected from the group of: (a) the DNAsequence having a heavy chain encoding sequence corresponding to asequence of FIG. 35 (SEQ ID NO: 8, 81) and a light chain encodingsequence corresponding to a sequence of FIG. 36 (SEQ ID NO: 10, 82); (b)the DNA sequence having a heavy chain encoding sequence comprising theheavy chain variable region CDR1, CDR2 and CDR3 sequences as set out inFIG. 35 and SEQ ID NO: 8 or 81, and a light chain encoding sequencecomprising the light chain variable region CDR1, CDR2 and CDR3 sequencesas set out in FIG. 36 and SEQ ID NO: 10 or 82; (c) DNA sequences havinga heavy chain and light chain encoding sequence comprising the heavychain variable region CDR1, CDR2 and CDR3 sequences as set out in FIG.35 and SEQ ID NO: 8 or 81, and a light chain encoding sequencecomprising the light chain variable region CDR1, CDR2 and CDR3 sequencesas set out in FIG. 36 and SEQ ID NO: 10 or 82 that hybridize to the DNAsequence of subpart (a) or (b) under standard hybridization conditions;and (d) DNA sequences that code on expression for an amino acid sequenceencoded by any of the foregoing DNA sequences.
 9. A recombinant DNAmolecule comprising a DNA sequence of claim
 8. 10. The recombinant DNAmolecule of claim 9, wherein said DNA sequence is operatively linked toan expression control sequence.
 11. The recombinant DNA molecule ofclaim 10, wherein said expression control sequence is selected from thegroup consisting of the early or late promoters of SV40 or adenovirus,the lac system, the trp system, the TAC system, the TRC system, themajor operator and promoter regions of phage λ, the control regions offd coat protein, the promoter for 3-phosphoglycerate kinase, thepromoters of acid phosphatase and the promoters of the yeast-matingfactors.
 12. A unicellular host transformed with a recombinant DNAmolecule of claim 9 or
 10. 13. The unicellular host of claim 12, whereinthe unicellular host is selected from the group consisting of E. coli,Pseudomonas, Bacillus, Streptomyces, yeasts, CHO, R1.1, B-W, L-M, COS 1,COS 7, BSC1, BSC40, and BMT10 cells, plant cells, insect cells, andhuman cells in tissue culture.
 14. A recombinant virus transformed withthe recombinant DNA molecule, or a fragment thereof, in accordance withclaim 9 or
 10. 15. A vector which comprises the recombinant DNA moleculeof claim 9 or
 10. 16. The vector of claim 15, wherein the expressioncontrol sequence comprises a bacterial, yeast, insect or mammalianpromoter.
 17. The vector of claim 15, wherein the vector is a plasmid,cosmid, yeast artificial chromosome (YAC), bacteriophage or eukaryoticviral DNA.
 18. A host vector system for the production of a polypeptidewhich comprises the vector of claim 15 in a suitable host cell.
 19. Thehost vector system of claim 18, wherein the suitable host cell comprisesa prokaryotic or eukaryotic cell.
 20. A method of obtaining apolypeptide in purified form which comprises: (a) introducing the vectorof claim 15 into a suitable host cell; (b) culturing the resulting hostcell so as to produce the polypeptide; (c) recovering the polypeptideproduced in step (b); and (d) purifying the polypeptide so recovered instep (c).
 21. A nucleic acid probe capable of screening by hybridizationfor the antibody sHIgM22, a recombinant antibody derived therefrom, or amonomer or an active fragment of sHIgM22 wherein said nucleic acid probeis prepared from and comprises sequence from the DNA sequence of claim8.